In recent years, the number of genome-wide association studies (GWAS) carried out for various economically important animal traits has been increasing. GWAS discoveries provide summary statistics that can be used both for targeted marker-oriented selection and for studying the genetic control of economically important traits of farm animals. In contrast to research in human genetics, GWAS on farm animals often does not meet generally acceptedstandards . Thisgreatly complicates the use of GWAS results for breeding needs. Within the framework of human genetics, there areseveral technological solutions for researching the harmonized results of GWAS, including one of the largest, theGWAS-MAP platform. For other types of living organisms, including economically important agricultural animals,there are no similar solutions. To our knowledge, no similar solution has been proposed to date for any of the speciesof economically important animals. As part of this work, we focused on creating a platform similar to GWAS-MAP forworking with the results of GWAS of sheep, since sheep breeding is one of the most important branches of agriculture.By analogy with the GWAS-MAP platform for storing, unifying and analyzing human GWAS, we have createdthe GWAS-MAP|ovis platform. The platform currently contains information on more than 34 million associations betweengenomic sequence variants and traits of meat production in sheep. The platform can also be used to conductcolocalization analysis, a method that allows one to determine whether the association of a particular locus withtwo different traits is the result of pleiotropy or whether these traits are associated with different variants that are inlinkage disequilibrium. This platform will be useful for breeders to select promising markers for breeding, as well asto obtain information for the introduction of genomic breeding and for scientists to replicate the results obtained. A genome-wide association study (GWAS) is moderntechnique to detect genome loci associated with differenttraits both quantitative and binary ones. GWASs have beenwidely used in human and animal genetics . Since 2007, an annual increase of GWAS-basedinvestigations to search for trait-associated loci has beenobserved. A GWAS results in the so-called summary statistics,a text tables where each line is a result of associationanalysis between a studied trait and a single nucleotidepolymorphism (SNP). In breeding, such summary statisticscan be used to devise marker-assisted and genomic selectionmodels and identify target candidates for genomeediting. Thanks to the new-generation technologies ofgenome-wide genotyping and next-generation sequencing,modern GWASs enable one to study hundreds of thousandsand oftentimes millions of SNP associations with dozensof traits in huge samples including some tens thousandsof animals. In other words, GWAS summary statistics arehuge datasets , whose processing isfar from being trivial and requires, apart from their collection,a platform to provide proper quality control, storage,access and processing tools.In the field of human genetics, such a platform, knownas GWAS-MAP , serves for unification,storing and analysis of millions of associations forthousands of human traits. However, to our knowledge,there is still no such a solution even for a single kind oflivestock farming. There are only databases to processquantitative trait loci significantly associated with studiedtraits. The biggest of such databases is AnimalQTLdb thatcontains information on quantitative-trait loci for differentkinds of livestock . There are similarsolutions for particular kinds of animals such as the iSheepdatabase that provides access to the known quantitative-traitloci, genotyping and sequencing data of sheep . Since none of the mentioned solutions providesopportunities for storing, unification and analysis of GWASsummary statistics, the objective of the presented study wasto create such a platform for sheep.Sheep breeding is an important branch of animal farmingwith mutton being its most popular product. Since 2013,more than 20 GWASs of economically important sheeptraits have been known. Here, S. Bolormaa et al. (2016)is of particular note being the biggest GWAS for a meatproductivity traits in sheep. The authors investigated acohort of more than 10,000 animals to study informationon 56 different genetic traits related to meat productivityand found more than 70 significantly associated SNPsincluded in 23 loci.Here we present the GWAS-MAP|ovis platform that isanalogous to GWAS-MAP and can be used for aggregation,unification, storing and analysis of the GWAS summarystatistics of the economically important traits affectingmeat production in sheep. Currently, the platform containsmore than 34 mln associations for 80 such traits. Breedersmay find it useful for selection based on the effects of thealleles associated with studied traits and on perspectiveselection markers. It may also be useful for the scientistsinvestigating sheep genetics. In particular, the platformenables for GWAS quality control; search for significantlyassociated SNPs; GWAS results visualization and colocalizationanalysis.Developing the GWAS-MAP|ovis platform. GWAS resultsare commonly summary statistics being big text tablesin which a single line is the results of association analysis to match a studied trait and SNP. Summary statistics caninclude different columns such as SNP identifier (if any);SNP position in a physical genome map (chromosome andposition), SNP imputation or genotyping quality controlmetrics; effective and reference alleles; effective allele fre-quency; SNP effect estimation; standard error and p- value;sample size and other technical variables specific forparticular research. The minimum set of the variablesnecessary for summary statistics processing includes anSNP identifier, effective and reference alleles, effect estimationand a standard error. Without loss of information,the standard error can be replaced by either the p-value orby z-statistic. Since the number of SNPs is huge, the sizeof the file containing a summary statistics table can reachseveral gigabytes in a case the number of characterizedSNPs exceeds 20 millionThe GWAS-MAP platform  to store GWAS results in the humanwas used as a basis to design the solution in question. Forpresentation and visualization of genetic data, the PheLiGeweb interface was introduced .The flow chart of GWAS-MAP|ovis can be seen in Fig. 1.To store and process data, GWAS-MAP uses two databasemanagement systems, ClickHouse and PostgreSQL.While ClickHouse serves to store matched summary statistics,PostgreSQL is used to store the metadata describing thesources summary statistics come from, a GWAS dataset anddata analysis features. They may include e. g., a reference tothe publication data come from; trait name; description ofthe model used for analysis; the number of studied animals,etc. Their full list is available in Supplementary material 11.The two systems are deployed in an LXC container andoperate using an Ubuntu 18.04 operating system image.One of the key steps in setting up the database was creatinga linkage disequilibrium catalogue and the so-calledreference table (see the description below). To provide convenient access to the database and enable data visualizationas interactive graphs, the PheLiGe-ovis web interface wasdeployed in another LXC container and operated using thesame operating system image. The web interface is availableat https://pheligeovis.icgbio.ru/.http://vavilov.elpub.ru/jour/manager/files/Suppl_Kirichenko_engl.pdfSupplementary Materials are available in the online version of the paper:The designed platform having two databases to storesummary statistics and metadata and a web interface wascalled GWAS-MAP|ovis and is available via public (web)and private (ssh) interfaces. The general principles of howto operate its web interface and main tools are describedin the on-line assistant program, whose window pops upwhen pressing the red button under the Associations menuitem in the top right corner of the platform’s main page.Linkage disequilibrium catalogue is an SNP list thatcontains the following information for each included SNP:identifier (rs_id); chromosome and position (base on theOAR_V3.1 genome assembly); effective and referenceallele, and effective allele frequency in referent population.To create the catalog were used the genome data ofa reference population (N = 96) including 18 Romanovbreed sheep, 6 Katahdin sheep, 10 argali, 48 F1 hybridsfrom crossing F1  and Romanovbreeds, and 14 F1 hybrids from crossing Romanov andKatahdin breeds. All the animal had been genotyped usingInfinium® HD SNP Bead-Chip  and used for replication inour recent paper . Preparing the referencetable involved removing all duplicated and monomorphicSNPs; the alleles were placed in lexicographicorder; sex chromosome data were removed. For the timebeing, the disequilibrium catalogue contains informationon 523.578 SNPs. Their autosomal distribution can befound in Supplementary material 2 and their minor allelefrequencies in Supplementary material 3.The linkage disequilibrium catalogue is files containingthe genotypes of the reference population animals forevery SNP included in the reference table. The files are inbinary format PLINK . The catalogueis used to calculate the linkage disequilibrium betweenSNPs for finding a proxy SNP in the PheLiGe-ovis webinterface.Module of unification and quality control GWAS results.This module is used to download summary statisticsin the database. Since these files are obtained from differentsources and often have different formats, they have to beunified first. At this stage, matches between rs_id and theposition in the chromosome and between the effective andreference alleles are checkedAs the next stage, the summary statistics pass qualitycontrol that includes three stages: (1) comparison of GWASallele frequencies against those in the linkage disequilibriumcatalogue; (2) checking effect-size distribution,and (3) verifying if the p-value corresponds to z-statistic(effect estimation divided by a standard estimation error). The module generates an html report that can be used todetect unification and quality-control errors. If data passthe control, the summary statistics are stored in the platform’sdatabase.Content managementTo fill the database, a search for published summarystatistics was carried out in an early published databaseof the QTL and genes associated with meat productivitytraits in sheep . Out of 46 publicationsanalyzed, only S. Bolormaa et al. (2016) provided accessto genome-wide summary statistics. Before being loadedin the GWAS-MAP|ovis database, all the data were unifiedand they passed the quality control. As a result, 488,074associations for each of the 56 traits listed in the paperwere downloaded inIn addition to the data provided by S. Bolormaa et al.(2016), the GWAS-MAP|ovis database also contains thesummary statistics obtained from the Russian sheep population(N = 50–108) early used for replication analysis . This summary statistics described 7 compositeindices related to the meat productivity measured for threedifferent time periods  and ananimal’s weight also measured for three time periods . As a result,the platform’s database has accumulated information onmore than 34 million SNP associations for 80 differenttraits related to meat productivity in sheep. The availabledataset and its short description are presented in the TablePleiotropy analysisOne of the advantages of the designed platform is that itcan be used to perform colocalization analysis (SMR-𝜃), which enables one to comparethe association patterns of a particular genome locus fordifferent traits and make conclusions whether this locushas a pleiotropic effect on a certain trait. In a nutshell, inpresence of this effect, the ratio of estimated SNP effects ontwo traits in a studied locus is expected to be insignificant.However, if the association patterns are different i. e., theeffect ratio changes significantly from one SNP to another, it is most likely that the locus contains different functionalpolymorphisms for each of the traits and they are being inlinkage disequilibrium.Statistic 𝜃 is a weighted correlation, whose computationrequires information on 𝑝-values and an effect direction.The high absolute value  means a locus has apleiotropic effect on investigated traits. If 𝜃 is positive, theSNPs of the locus have a similar effect direction towardsthe traits, and a divergent one if negative. An example ofcolocalization analysis is presented in Fig. 2.In the framework of the presented study, colocalizationanalysis was carried for the most significantly associatedSNP of the LCORL1 gene in the data by . For SNP rs401834107, 12 traits of genome-widesignificance level  associated with thislocus were indicated. The SMR-𝜃 method was applied tocompare association patterns for all the 12 traits, the resultsare demonstrated in Fig. 3. The heat map characterizing𝜃-values shows the traits form two clusters with differenteffect correlation directions, which means the locus had apleiotropic effect inside each of the clusters. At the sametime, the same strong effect was observed between traitsfrom different clusters with divergent SNP effect on thetraits . It is noteworthy that the slaughterweight trait was a dropout since some of its 𝜃-values turnedout to be low (possible pleiotropy absence). However, dueto the pleiotropic effect the locus had on the other traits,the low 𝜃-values in this particular case can be consideredeither as a statistic artifact or as a value produced due toinsignificant analysis capacity.Thus, it has been found that locus rs401834107 had apleiotropic effect on at least 12 different traits associatedwith meat production in sheep. The presented data is anexample of a first in the world colocalization analysisperformed using sheep GWAS results.Searching for candidate DNA markersfor marker-assisted selectionIn addition to the described pleiotropy analysis, anotheradvantage of the GWAS-MAP|ovis platform is that it allowsone to use previously loaded summary statistics tosearch for candidate markers to perform marker-assistedselection (MAS). In the presented study, such a search for the hot-carcass-weight (HCWT) trait was performed to estimatethe markers’ predictive potential in a Russian sheepcohort. To begin with, a clumping procedure was carriedout. To do this, the private (ssh) interface was used to selectthe summary statistics of interest and set up their significantthreshold of p-value < 1E–07. Four independent lociwere detected . A table containing information on SNPwith the highest associations for each of the loci, theirchromosomes, positions, effector and reference alleles andp-values was formed for further analysis.The found loci were used to estimate the breeding value(BV) of 94 animals  to be the backcrosshybrids of Romanov and argali sheep. Their BV wasestimated as:where b is the effect of the effective allele of the most associatedSNP in every locus; g is the genotypes of 94 animalsfor that SNP encoded as 0, 1, 2 in relation to the effectiveallele’s quantityCases then underwent linear regression to find how significantthe estimated BVs were for predicting a species’phenotype. As a trait, an animal’s weight in six days afterbirth was considered. The used model was p ~ BV + cov,where p is a phenotype’s value; cov is the covariates includinginformation about animal gender, yield number, and twofirst main components of their kinship matrix. As a result,the estimated BV was found to be significantly associated with the trait, which confirmed that theselected loci had a significant effect on an animal’s weightin six days after birth.In addition to the performed analysis, it was also estimatedwhat body weight increase could be expected in theanimals whose BV was included in the fourth (top) quartile(> 75 %). Student’s t-criterion was used to compare themean values of the phenotype corrected for the abovementionedcovariants for the whole cohort and the animalsincluded in top BV quartile. It was found that the weightof the top-quartile animals was 1.8 %  higher than that in the averaged population.Thus, the selected markers can be potentially used fordevelopment of MAS test systems in sheep.The GWAS-MAP|ovis platform has been designed foraggregation, unification, storage and analysis of GWASsummary statistics in sheep. Today, it contains more than34 million SNP associations for 80 different traits relatedto meat productivity in sheep. The platform enables oneto search for information on associations for a particularSNP, plot Manhattan graphs and carry out colocalization analysis. The platform can be useful for scientists as aninvestigating tool to study sheep genetics and for breedersas a solution for searching for potential MAS loci.The authors declare no conflict of interest.Bolormaa S., Hayes B.J., van der Werf J.H.J., Pethick D., GoddardM.E., Daetwyler H.D. Detailed phenotyping identifies geneswith pleiotropic effects on body composition. BMC Genomics.2016;17:224. DOI 10.1186/s12864-016-2538-0.Hu Z.-L., Park C.A., Wu X.-L., Reecy J.M. Animal QTLdb: an improveddatabase tool for livestock animal QTL/association datadissemination in the post-genome era. Nucleic Acids Res. 2013;41(D1):D871-D879. DOI 10.1093/nar/gks1150.Momozawa Y., Dmitrieva J., Théâtre E., Deffontaine V., Rahmouni S.,Charloteaux B., Crins F., Docampo E., Elansary M., Gori A.- S.,Lecut C., Mariman R., Mni M., Oury C., Altukhov I., Alexeev D.,Aulchenko Y., Amininejad L., Bouma G., Hoentjen F., LöwenbergM., Oldenburg B., Pierik M.J., van der Meulen-de Jong A.E.,van der Woude C.J., Visschedijk M.C., International IBD Genetics Consortium, Lathrop M., Hugot J.-P., Weersma R.K., De Vos M.,Franchimont D., Vermeire S., Kubo M., Louis E., Georges M. IBDrisk loci are enriched in multigenic regulatory modules encompassingputative causative genes. Nat. Commun. 2018;9(1):2427.DOI 10.1038/s41467-018-04365-8.Shashkova T.I., Aulchenko Y.S. Database for storing and quickly accessingthe results of genome-wide and regional association studies.Patent RF No. 2020620869. 2020. (in Russian)Shashkova T.I., Gorev D.D., Pakhomov E.D., Shadrina A.S., SharapovS.Z., Tsepilov Y.A., Karssen L.C., Aulchenko Y.S. TheGWAS-MAP platform for aggregation of results of genome-wideassociation studies and the GWAS-MAP|homo database of 70 billiongenetic associations of human traits. Vavilovskii ZhurnalGenetikii Selektsii= Vavilov Journal of Genetics and Breeding.2020;24(8):876-884. DOI 10.18699/VJ20.686.Shashkova T.I., Pakhomov E.D., Gorev D.D., Karssen L.C., Joshi P.K.,Aulchenko Y.S. PheLiGe: an interactive database of billions ofhuman genotype-phenotype associations. Nucl. Acids Res. 2021;49(D1):D1347-D1350.Visscher P.M., Wray N.R., Zhang Q., Sklar P., McCarthy M.I.,Brown M.A., Yang J. 10 years of GWAS discovery: biology, function,and translation. Am. J. Hum. Genet. 2017;101(1):5-22. DOI10.1016/j.ajhg.2017.06.005Wang Z.-H., Zhu Q.-H., Li X., Zhu J.-W., Tian D.-M., Zhang S.-S.,Kang H.-L., Li C.-P., Dong L.-L., Zhao W.-M., Li M.-H. iSheep:an integrated resource for sheep genome, variant and phenotype.Front. Genet. 2021;12:714852. DOI 10.3389/fgene.2021.714852.Zlobin A.S., Nikulin P.S., Volkova N.A., Zinovieva N.A., IolchievB.S., Bagirov V.A., Borodin P.M., Aksenovich T.I., TsepilovY.A. Multivariateanalysis identifies eight novel loci associatedwith meat productivitytraits in sheep. Genes. 2021;12(3):367. DOI 10.3390/genes12030367.Zlobin A.S., Volkova N.A., Borodin P.M., Aksenovich T.I., TsepilovY.A. Recent advances in understanding genetic variants associatedwith growth, carcass and meat productivity traits in sheep(Ovis aries): an update. Arch. Anim. Breed. 2019;62(2):579-583.DOI 10.5194/aab-62-579-2019.


Polyolefins, including polyethylene (PE), polypropylene (PP) and polystyrene (PS), are widely used plastics in our daily life. The excessive use of plastics and improper handling methods cause considerable pollution in the environment, as well as waste of energy. The biodegradation of polyolefins seems to be an environmentally friendly and low-energy consumption method for plastics degradation. Many strains that could degrade polyolefins have been isolated from the environment. Some enzymes have also been identified with the function of polyolefin degradation. With the development of synthetic biology and metabolic engineering strategies, engineered strains could be used to degrade plastics. This review summarizes the current advances in polyolefin degradation, including isolated and engineered strains, enzymes and related pathways. Furthermore, a novel strategy for polyolefin degradation by artificial microbial consortia is proposed, which would be helpful for the efficient degradation of polyolefin.  Plastics are one of the most widely used and vital materials in the modern world. However, the enormous manufacture and abuse of plastics place a huge burden on the environment. It was reported that about 58% of plastic waste was placed in landfills or discharged directly into the environment, 24% was burned and only 18% of plastic waste was recycled globally  and are Rhodococcus ruber C208, which adhered to PE films and utilized LDPE films as the sole carbon source. Later, researchers found that R. ruber C208 could secrete laccase in vitro and that laccase also play a vital role in PE degradation [Azotobacter beijerinckii HM121, was reported to degrade PS [It was reported that the degradation rate of PP and PS was improved after pretreatment by UV and high temperature . Thereforadation , soybeanradation  and alkaradation ,12 are agrade PS . Moreovegrade PS ,15. Manygrade PS ,18,19,20This review summarizes natural strains, enzymes and engineered microbial chassis for the biodegradation of polyolefins. Based on the former content, we provide an idea for polyolefin degradation by artificial microbial consortia and discussed the prospect.Polyolefins, including PE, PP and PS, are composed of C–C and C–H bonds, which are more stable against degradation than ester bonds. During polyolefin biodegradation, C–C and C–H bonds are oxidized. Many types of microorganisms have been isolated from sea water, compost and activated sludge with the capacity for polyolefin biodegradation. The oxidation of polyolefins can be divided into four stages, including biodeterioration, biofragmentation, bioassimilation and mineralization . As showNumerous of microorganisms with the capacity for polyolefin degradation, including bacteria, fungi and microbial consortia, have been isolated from the environment, such as soil containing plastic waste, the ocean and the guts of plastic-eating worms. These microorganisms are capable of utilizing polyolefins as sole carbon source or can generate depolymerases involved in polyolefin degradation. Therefore, the screening of these microorganisms is vital for the further degradation of polyolefins. Polyolefin-degrading bacteria can be identified by the following procedure. Soil samples collected from an area containing polyolefin waste was mixed with water and shaken . In ordeR. ruber [Pseudomonas [Bacillus [Acinetobacter [R. ruber C208, one of the most efficient bacteria for PE biodegradation, is a Gram-positive bacterium isolated from soil and is capable of degrading unpretreated LDPE at a rate of 0.9% per week [R. ruber C208 [R. ruber C208 is also capable of degrading PS, achieving a weight loss of 0.8% after 8 weeks [R. rhodochrous ATCC 29672 exhibited the ability to degrade PP based on the characterization of changes in the metabolic activity of bacteria, such as ATP content, ADP/ATP ratio and cell viability [At present, most single strains that are capable of degrading polyolefins are bacteria, which can form biofilms on the surface of polyolefins or destroy the surface of polyolefins. R. ruber ,25,26, Pudomonas ,27,28, BBacillus ,29, Acintobacter  etc. R. per week . An incrper week . Changesiability .Aspergillus clavatus JASK1, Phanerochaete chrysosporium NCIM 1170, Engyodontium album MTP091 and Curvularia sp. isolated from landfill soil have also been shown to play an important role in PE biodegradation [As shown in radation ,36,37. 2 measurement [Biodegradation should not only be determined by weight loss. Chemical properties, as well as changes in Mw (molecular weight) and Mn (molecular number), which can be characterized Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC) analyses, are also vital for the measurement of the degree of polyolefin degradation. After the biodeterioration stage, the nature and occurrence of functional groups on the surface of polyethylene substrates are changed, which can be studied by FTIR  spectrossurement . FurtherEnterobacter asburiae YT1 and Bacillus sp. YP1 were isolated from the guts of waxworms with the capability of degrading PE. Over 28-day incubation of the two strains on PE films, the physical properties (tensile strength and surface topography), chemical structure (hydrophobicity and appearance of carbonyl groups), Mw (accompanied by the formation of daughter products) and weight loss were detected [Pseudomonas aeruginosa PAO1 with LDPE films. After exposure to P. aeruginosa PAO1, the LDPE sample turned into a mixture of long-chain fatty acids, esters, hydrocarbons, oxygenated chemical compounds predominantly containing aldehydes, ketones, esters and ether groups, unsaturated fatty acids and certain unknown compounds. Given that the structure of polyolefins is similar to that of alkanes, some strains with an ability to degrade alkanes also have an effect on PE degradation. Alcanivorax borkumensis, a bacterial strain isolated from the sea that can utilize alkane as carbon source [As previously reported, some waxworms can chew and eat plastics ,42,43,44detected . Kyaw etdetected  incubaten source  was founn source .Bacillus sp. and two Paenibacillus sp. was isolated from a landfill site. After incubation with the mixed microbial consortium for 30 days, the weight and the mean diameter of the PE sample were reduced 16.7% and 22.8%, respectively [Lysinibacillus xylanilyticus and Aspergillus niger was found to be capable of degrading PE [Brevibacillus sps. and Aneurinibacillus sp. from waste landfills. During a 140-day incubation of the two strains on PE samples, weight loss for LDPE and HDPE strips reached 58.2% and 46.6% respectively, and weight loss for LDPE and HDPE pellets were 45.7% and 37.2%, respectively. As with a single bacterium, UV pretreatment aided in the degradation of polyolefin samples. In addition, the result of GC-MS analysis indicated that PE samples were degraded into cis-2-chlorovinylacetate, tri-decanoic acid and octadecanoic acid. Muenmee et al. [4:40% CO2) and a microbial consortium composed of Methylocyctis sp., Methylocella sp., Methylobactor sp., Methyloccus capsulatus., Nitrosomonas sp., Nitrosomonas europaea, Nitrobacter winogradskyi, Nitrobacter hamburgensis, Burkholderia sp., Pseudomonas sp. and Xanthobacter sp. After a reaction period of 3 months under semi-aerobic landfill conditions where different aeration rates were supplied, the plastic samples were degraded into hydrocarbon and oxygenated compounds, such as aliphatic alkanes, alkenes, alcohols and esters. The degradation products of different plastic types, such as the degraded products of HDPE, are mainly alkanes , alkenes  and some alcohols  were found, while for LDPE, only one alcohol (C11H24O) was found.In addition to single bacteria, there are many microbial consortia that are capable of degrading PE isolated from various environments . A mixedectively . A microading PE . After Uading PE  isolatedAzotobacter beijerinckii HM121. However, no PP-degrading enzymes have been identified to date. The result of enzymatic degradation of PE is that oxidation groups are introduced into the PE chain, which means polyolefins cannot be oxidized into monomers as the only act of these enzymes. Most PE-degrading enzymes can only perform terminal oxidation  and subterminal oxidation  of PE. For example, laccase and manganese peroxidase can perform terminal oxidation, and the AlkB family can degrade n-alkanes, the main component of polyethylene, through either terminal or subterminal hydroxylation reactions [Most the enzymes capable of degrading PE are oxidoreductases. As shown in eactions . TherefoR. ruber C208 with the ability to degrade LDPE. As laccases contain four copper ion bonding sites, copper markedly affects their induction and activity, resulting in PE degradation. mRNA quantification by RT-PCR revealed a 13-fold increase in laccase mRNA levels in copper-treated cultures compared with an untreated control. The addition of copper to C208 cultures containing PE enhanced the biodegradation of PE by 75% [Trichoderma viride fungus and 1-HBT to degrade LDPE. After incubation for 10 days, the weight loss of LDPE came to 2.3% [Laccases (EC 1.10.3.2), which belong to the so-called blue-copper family of oxidases, can catalyze the oxidation of a wide range of phenols and arylamines. Laccases, which are glycoproteins, have been reported in higher plants, fungi and bacteria. A laccase was purified from E by 75% . A laccaE by 75% . Some reE by 75% . JohnnieE. coli, which means that E. coil is a good chassis for the heterologous expression of P450 genes. P450 enzymes can identify multiple substrates and catalyze diverse reactions, such as C–H hydroxylation; C=C double-bond epoxidation; heteroatom oxygenation; O-, N- and S-dealkylation; aromatic coupling; and C–C bond cleavage [Cytochrome P450 , a member of a superfamily of heme–thiolate proteins, is distributed in most living organisms. There are more than 300,000 P450 genes. However, no P450 genes were found in cleavage . BecausePseudomonas species isolated from oil-contaminated areas, is a membrane-bound, non-heme di-iron monooxygenase [Alkane, which is composed of C–C bonds and C–H bonds, has a similar structure to that of PE. Therefore, alkane mono-oxygenase enzymes are potential candidates for the degradation of PE. One alkane hydroxylase, namely AlkB, has been reported to degrade PE . AlkB, wxygenase . P. chrysosporium. Manganese peroxidase was first identified as a lignin-degrading enzyme. It was reported that the addition of Mn (II) to nitrogen- or carbon-limited culture medium enhanced PE degradation [Manganese peroxidase was purified from a lignin-degrading fungus: radation . E. coli and Y. lipolytica, have been applied to the expression and secretion of polyolefin-biodegrading enzymes to date [As shown in  to date ,78,79. TE. coli:E. coli is one of the most widely used model microorganisms for production of recombinant proteins. As a model microorganism, E. coli has advantages in many aspects, such as a simple genetic background, ease of genetic modification and simple growth conditions. Engineered E. coli has been used to express alkane hydroxylase to degrade LMWPE. The alkB gene, which was cloned from Pseudomonas sp. E4, was introduced into E. coli BL21. After incubation for 80 days at 37 °C with engineered E. coli, 19.3% of the LMWPE was degraded [alkB with its coenzyme genes—rubA1, rubA2 and rub—in E. coli BL21. The result indicated that 30.5% of the carbon of LMWPE-1 degraded into CO2 after 78 days [Acinetobacter johnsonii JNU01 degraded PS, and this finding was later confirmed by recombinant alkB in E. coli BL21 [alkB2 in E. coli, and the result indicates that alkB2 was more efficient for low-molecular-weight PE biodegradation than alkB1 [E. coli and showed PE degradation ability [degraded . The ruboli BL21 . These ran alkB1 . A lacca ability .E. coli can be used for laccase heterologous expression and secretion [E. coli is that it is easy for E. coli to form inclusion bodies when expressing extracellular enzymes. Mo et al. [Trametes versicolor and bacterial BPUL laccases from Bacillus pumilus for the purpose of degrading β-estradiol. The result of Western blot analysis indicates that laccase was detected both in vivo and in vitro in E. coli, which means that some laccase stayed in inclusion bodies instead of being secreted in vitro. Given that laccase has been widely used for the biodegradation of PE [E. coli is a potential method for the biodegradation of PE. Many recent studies have shown that engineered ecretion ,83,84. Iecretion  expresseo et al.  expresseon of PE ,75,85,86Yarrowia lipolytica:Y. lipolytica is a Crabtree-negative ascomycete yeast with good protein secretion capacities. Compared to other yeasts, Y. lipolytica lacks α-1,3-mannosyltransferase, a factor that limits the amount of excessive mannosylation of secreted heterologous glycoproteins and constitutes a valuable asset for the production of therapeutic proteins [Y. lipolytica W29 is a wild-type strain with a remarkable characteristically high secretion level of proteins [Y. lipolytica W29 ura302 was obtained through genetic convention of URA3 into ura3-302 in Y. lipolytica W29, and it was able to utilize sucrose and molasses as a carbon source under the control of XPR2 promoter. After the genetic convention of XPR2 into xpr2-322 and AXP1 into axp1-2, which indicates inactivation of alkaline extracellular protease and acid extracellular protease that would degrade foreign extracellular protein, a strain with high heterologous protein production capability called Y. lipolytica Po1f was obtained. A new strain called Y. lipolytica Po1g that carries a pBR322 docking platform was obtained through the integration of PINA300′ plasmid in Y. lipolytica Po1f [Y. lipolytica Po1g was induced with a YLEX kit for expression/secretion of heterologous proteins [Trametes versicolor was expressed in Y. lipolytica Po1g for the biodegradation of PE. Compared to the yeast secretion signal, the native secretion signal showed higher enzyme activity in the culture medium. The yield of laccase reached 2.5 mg/L (0.23 units/mL) [Y. lipolytica is a promising method of PE degradation.proteins . Y. lipoproteins . Y. lipoica Po1f . Y. lipoproteins . A laccanits/mL) . LaccaseB. amyloliquefaciens MT45 and B. amyloliquefaciens X82 improved substrate utilization and increased the product titer by 3.3-fold [B. subtilis, Rhodococcus jostii and P. putida to degrade PET. The result showed that the microbial consortium could degrade PET film, with weight loss reaching 23.2% under ambient temperature. The artificial microbial consortium successfully relieved the metabolic inhibition of TPA and EG [Microorganisms do not exist independently in natural environments, and they usually live in complex communities. They communicate through quorum sensing and share metabolites and enzymes, expanding the substrate range compared with single bacteria. Therefore, artificial microbial consortia can be constructed to degrade plastics that are not easily degraded naturally . The app3.3-fold . Another3.3-fold . In our A and EG ,97. SomeA and EG . As for Most microbial consortia with the ability to degrade polyolefins identified to date are natural microbial consortia. Skariyachan et al.  formulatMost single bacteria or microbial consortia isolated from nature can only degrade polyolefins into long-chain aliphatic compounds  at most. Few studies have achieved the goal of complete degradation of polyolefins. Building artificial microbial consortia provides a potential means for the complete degradation of polyolefins using polymer-degrading strains and long-chain aliphatic-compound-degrading strains. Combining polyolefin-degrading modules and long-chain aliphatic-compound-degrading module is promising means by which to completely degrade polyolefins . As showAspergillus sp. [Bacillus sp. [Yarrowia sp. [In contrast to polyolefins, long-chain aliphatic compounds can transfer into cells through active transport, passive transport, endocytosis and free diffusion and be metabolized in the cells. Many researchers have reported microorganisms isolated from environments that are capable of degrading alkanes, such as llus sp. , Bacillullus sp.  and Yarrowia sp. . Many enowia sp. . As for owia sp. . Alkenesowia sp.  and thenConstructing an artificial microbial consortium that includes part or all of the following strains is a vital metabolic strategy to solve the current problems of polyolefin degradation: polyolefin degraders, long-chain aliphatic-compound degraders and biosurfactant producers. Artificial microbial consortia can perform more complex tasks compared to pure strains, and the division of labor is clearer than that of artificially formulated consortia.In this review, we summarized the current advances in polyolefin biodegradation from pure natural strains, natural microbial consortia, enzymes, engineered chassis and artificial microbial consortia. The prospect of the biodegradation of polyolefins by artificial microbial consortia was also discussed. Constructing artificial microbial consortia is a promising strategy for the biodegradation of polyolefin. The use of artificial microbial consortia is a promising method for the degradation of polyolefins and long-chain aliphatic compounds. Compared to natural microbial consortia, the division of labor in artificial microbial consortia is clearer and more favorable for follow-up studies. The possibility of building an artificial microbial consortium including a biosurfactant producer, a polyolefin degrader, an alkene degrader and an alkane degrader was proposed in this review. There have been many recent advances in synthetic biology and metabolic engineering ,110,111.


Plasmodium malariae is a neglected human malaria parasite with low parasitemia that often results in the misdiagnosis and underestimation of the actual disease burden of this pathogen. Microscopy is the best diagnostic tool, despite the fact that rapid diagnostic tests (RDTs) are the best surveillance tool for malaria diagnosis in many rural areas for their ease of use in elimination settings. For parasite antigen detection other than P. falciparum, RDTs depend on essential glycolytic Plasmodium proteins, i.e., Plasmodium lactate dehydrogenase (pLDH) and Plasmodium aldolase (pAldo) antigens. There is a lack of species-specific test kits for P. malariae, and overall, its rapid antigenic test accuracy is questionable. False negative results can accelerate the burden of asymptomatic malaria infection and transmission. Here, we report a case of a malaria patient in Bangladesh infected with P. malariae who tested negative on pLDH and pAldo based RDTs. This case provides useful information for health providers to be aware of possible RDT failure and also for the future development of analytically sensitive test kits for P. malariae. Plasmodium malariae is one of the six parasites causing human malaria. Because of its low parasitemia and lower pathogenicity, it is often neglected. This protozoan parasite has been observed in all malaria-prone regions of the world [P. falciparum is the dominant and most relevant species, but P. malariae is present as well [P. malariae in the Chittagong Hill Tracts, but this parasite was documented in 9.5% of all malaria-positive asymptomatic participants [Plasmodium antigens, such as P. falciparum histidine-rich protein-2 (PfHRP2) for the diagnosis of P. falciparum only, and Plasmodium lactate dehydrogenase (pLDH) for the diagnosis of all Plasmodium species (pan-specific) or specifically P. falciparum (PfLDH) and/or P. vivax (PvLDH). Moreover pan-specific Plasmodium aldolase (pAldo) RDTs are available. However, none of the antigen detection kits is specific for P. malariae, P. ovale spp., or P. knowlesi [P. knowlesi is endemic, it remains problematic to distinguish P. malariae from P. knowlesi due to their similar morphology [he world . In Bang as well ,3,4,5. Aicipants . Rarely,icipants . Moreoveicipants . Speciesicipants . For theicipants . Malariaknowlesi . Today, rphology . Hence, P. malariae that tested negative on RDT (based on PfHRP2/pLDH) in Bangladesh despite the presence of a relatively high parasitemia. In this case, we report an infection with P. malariae. The initial parasite density count was 5380 P/µL. This initial count was performed by an experienced and WHO-certified microscopist. The patient received treatment in accordance with the national guideline [TM Rapid Malaria Pf/Pan , and (3) BinaxNOW® Malaria Test . The sample tested negative for PfHRP2  in Bandarban, Chittagong Hill Tracts, Bangladesh (92.3120° E longitude and 21.6523° N latitude). The patient resided near the AUHC and reported no travel activity in the past three months outside his territory. At the AUHC, the sample tested negative on RDT using a CareStart™ Malaria HRP2/pLDH (Pf/Pv) Combo  RDT. However, microscopical blood film examination confirmed infection with uideline . Three mr PfHRP2 a–c and nr PfHRP2 a,d.P. malariae was confirmed using 18S ribosomal RNA (rRNA) Plasmodium sp. and species-specific PCRs [P. malariae . On the other hand, a negative result for CareStart™ Malaria HRP2/pLDH (Pf/Pv) Combo  defined no cross reactivity of the sample with P. falciparum or P. vivax antigenic target  acts as a prognosis marker of disease severity in gression . In thisP. malariae to persist for a very long time in host facilities. Thus, circulating LDH antigen released by parasites assists in the formation of host antibodies [P. malariae [Studies have shown that PfHRP2 has a relatively long half-life (2–4 weeks), favoring the development of anti-PfHRP2 antibodies in the host blood and leading to false negative results The current authors also proposed that the similar blocking effect may hinder the performance of LDH or aldolase . A 72 hotibodies . This anmalariae .Plasmodium species, namely, P. falciparum and P. vivax. Neglected Plasmodium species like P. malariae are often overlooked. In our case, the RDT diagnosis of a symptomatic patient with relatively high parasitemia failed. It is evident that no reliable screening kits/RDTs are available for the diagnosis of P. malariae. Diagnosis with microscopy and PCR is often not possible in endemic rural settings. Since (mono-) infections of P. malariae are not uncommon, proper diagnosis may reveal more subclinical malaria cases in endemic countries. Lately, the paradigm shift from malaria control to malaria elimination in Bangladesh underscores the importance of having an efficient method of point-of-care testing for P. malariae. We recommend that the health care team in endemic areas be aware of RDT failings and propose that additional microscopical or molecular diagnostic tools might be needed to avoid false negative results. In endemic areas, malaria case management mainly focuses on the most relevant


Klebsiella pneumoniae. Furthermore, the cytotoxicity of the synergistic combinations was assessed in a human hepatocyte model. The results showed that the AgNPs (15–25 nm) were effective against Gram-negative bacteria (MIC of 16–128 µg/mL) but not Gram-positive strains (MIC of 256 µg/mL). Both wild-type and AMR K. pneumoniae had similar MIC values following exposure to AgNPs. Importantly, co-exposure to combinations of AgNPs and antimicrobial agents, including kanamycin, colistin, rifampicin, and vancomycin, displayed synergy against both wild-type and AMR K. pneumoniae isolates (except for vancomycin against AMR strain I). Notably, the tested combinations demonstrated no to minimal toxicity against hepatocytes. Altogether, this study indicates the potential of combining AgNPs with conventional antimicrobials to overcome AMR bacteria.The increasing prevalence of antimicrobial-resistant (AMR) bacteria along with the limited development of antimicrobials warrant investigating novel antimicrobial modalities. Emerging inorganic engineered nanomaterials (ENMs), most notably silver nanoparticles (AgNPs), have demonstrated superior antimicrobial properties. However, AgNPs, particularly those of small size, could exert overt toxicity to mammalian cells. This study investigated whether combining AgNPs and conventional antimicrobials would produce a synergistic response and determined the optimal and safe minimum inhibitory concentration (MIC) range against several wild-type Gram-positive and -negative strains and three different clinical isolates of AMR  Enterobacteriaceae as a priority 1  pathogen .,47.P. aeK. pneumoniae clinical isolates are presented in K. pneumoniae strains were sensitive to kanamycin but resistant to ciprofloxacin  were chosen for further testing (K. pneumoniae [+) [K. pneumoniae strains had similar MIC values following their exposure to AgNPs. This was also shown previously against different AMR bacterial strains, indicating the susceptibility of the AMR bacteria to AgNPs [+ [This study investigated the antimicrobial response of AgNPs against Gram-positive and -negative wild-type strains and showed that exposure to AgNPs resulted in significant antimicrobial activity against Gram-negative wild-type bacteria, including dermidis . Therefo testing . This aneumoniae ,50,51,52niae [+) ,53. The niae [+) . The obsniae [+) ,56. Impoto AgNPs ,58. SeveAgNPs [+ ,58,59.S. aureus, S. saprophyticus, S. sciuri, and S. epidermidis  of AgNPs against all tested Gram-positive bacterial strains, including dermidis . This fiectively . Another00 μg/mL . However00 μg/mL . For exa00 μg/mL . Additio strains ,53,62,63E. coli) strain and three AMR clinical isolates of K. pneumoniae using the checkerboard method and the fractional inhibitory concentration (FIC) index, as mentioned earlier. The FIC index is a mathematical expression used to evaluate the impact of drug combinations and determine whether synergy exists. For this study, the ΣFIC result was interpreted as synergism if the values were ≤0.5, indifference if values > 0.5 and ≤4, and antagonism if values > 4. The lowest ΣFIC value of each combination was considered a representative result for that combination [E. coli and AMR strains. Combinations of AgNPs with kanamycin or colistin against wild-type E. coli resulted in synergy, with remarkable ΣFIC values equal to 0.1   . The resK. pneumoniae. However, the findings reported in this study are consistent with the previous literature with regard to the development of synergy between AgNPs and the other antimicrobials against a broad range of bacterial strains. One key combination that produced consistent synergy against all AMR strains was AgNPs and kanamycin. Indeed, previous reports have demonstrated the effectiveness of this combination against a variety of bacterial strains [P. aeruginosa [E. coli, Salmonella enterica Serovar Typhimurium, and S. aureus at a percentage of ~95% compared to ~30% with kanamycin monotherapy [E. coli, P. aeruginosa, and AMR A. baumannii, with MIC values two- to fourfold lower than that of colistin alone [E. coli and AMR K. pneumoniae bacterial strains was AgNPs and rifampicin. The co-treatment with AgNPs increased the susceptibility of the bacteria to rifampicin, with a significant reduction in MIC values   . Such sydication . Howeverdication ,75,76,77+ (Trojan horse mechanism) [Enterobacteriaceae) against several antimicrobials, including kanamycin, colistin, and rifampicin [K. pneumoniae  using transcriptomics analysis identified oxidative stress and a triclosan-like antibacterial mechanism [As mentioned previously, the antimicrobial properties of AgNPs can be attributed to multiple different mechanisms, including disruption of the bacterial cell wall, generation of ROS, and internalization and release of Agchanism) ,28,29,30chanism) . Both kachanism) ,80. One chanism) ,67,81. Ifampicin ,83,84. Aechanism . AltogetStreptococcus mutans, Streptococcus oralis, and Aggregatibacter actinomycetemcomitans [P. aeruginosa, but not ciprofloxacin, as well as synergy in a AgNP-based combination with ampicillin against only one bacterial strain  among twelve other tested strains [Finally, the data in this study showed that the combinations of AgNPs and ampicillin (peptidoglycan cross-linking inhibitor) or ciprofloxacin (DNA gyrase inhibitor) produced no synergy against the tested bacterial strains (wild-type and AMR isolates). Previous studies demonstrated inconsistent findings for AgNP-based combinations with those two antimicrobials. One study showed that AgNP-based combinations rendered several pathogens susceptible to ampicillin and ciprofloxacin, including comitans . Other s strains ,86. Thes strains ,87.One critical aspect of newly developed therapeutic agents or materials is their human safety. Indeed, ensuring the safety of ENMs represents one of the major challenges in the field of nanotoxicology ,88. AgNPAgNPs  were purchased from US Research Nanomaterials, Inc. . Cation-adjusted Mueller–Hinton broth (CAMHB)  and Dulbecco’s Modified Eagle Medium (DMEM)  were used in the microbiology and cell culture studies, respectively. All antimicrobial agents used in this study were purchased from the Toronto Research Chemicals Company .The hydrodynamic size (nm), zeta potential (mV) (surface charge), and polydispersity index (PDI) of AgNPs were measured using a Zetasizer system  in deionized water, CAMHB, or DMEM. A transmission electron microscope  was used to confirm the size and the shape of the AgNPs.E. coli (ATCC 25922), Acinetobacter baumannii , K. pneumoniae (ATCC 13883), P. aeruginosa (ATCC 27853), Staphylococcus saprophyticus , Staphylococcus aureus , Staphylococcus sciuri , and Staphylococcus epidermidis . Three AMR K. pneumoniae clinical isolates were selected for checkerboard experiments based on their resistant phenotypes. These strains were isolated and collected from King Khalid University Hospital (KKUH), Riyadh, Saudi Arabia  , which included the following bacteria: i Arabia .The MIC can be defined as the lowest concentration of an antimicrobial agent that is required to inhibit the visible growth of a bacterium . The MICK. pneumoniae clinical isolates were prepared by inoculating colonies in 0.5 McFarland of CAMHB to produce 108 colony-forming units/mL (CFU/mL), followed by culture on blood agar and incubation overnight at 37 °C.All ATCC bacteria and AMR The presence of potential synergistic activities between AgNPs combined with other antimicrobial agents was evaluated by the checkerboard method against wild-type and selected AMR clinical isolates. The determination of the tested concentrations in the checkerboard depended on the MIC values. Two concentrations above the MIC value were chosen to account for the antagonistic effects, and the other chosen concentrations accounted for the synergistic and indifference effects. The general approach of the checkerboard assay resembles the microdilution method used to determine the MIC value. However, the two antimicrobials were diluted in two distinct directions to make the concentrations intersect with each other. Hence, AgNPs were diluted one way , while the other antimicrobial was diluted the other way , and then, the bacterial inoculum was added similarly to the MIC plate. Wells with untreated bacteria or medium were considered as positive and negative controls, respectively. Finally, the concentrations of the two agents that showed complete bacterial growth inhibition were recorded.FIC) index, a mathematical expression used to evaluate the impact of drugs’ combination and determine whether a synergistic effect exists or not [ΣFIC index was calculated using the following formula:Interpretation of the experimental results was conducted using the fractional inhibitory concentration  and 100 U penicillin/100 μg streptomycin/mL . Cell viability was determined by the MTT assay based on the cell mitochondrial activity. Cells were cultured in 96-well plates in serum-supplemented medium to ~80% confluency. Cells were then treated with AgNPs at final concentrations of 0.5–32 µg/mL in the presence or absence of antimicrobial combinations for 24 h in serum-free media. Working stocks of AgNPs were prepared from original stock (5 mg/mL) in sterile deionized water with careful and thorough mixing . MTT (500 μg/mL) was added to the cells, which were incubated at 37 °C and 5% CO2 until color development and crystal formation. Formazan crystals were then dissolved using isopropanol, and the absorbance was measured using a BioTek SynergyTM HT spectrophotometer at a wavelength of 570 nm . The cell survival rates were calculated and plotted as percentage viability using the relative absorbance equation as follows:HepG2 (ATCC HB-8065), a human liver cell model, was used to evaluate the safety of the AgNPs with/without the antimicrobials. The HepG2 cell line was purchased from ATCC . The HepG2 cells were cultured and propagated according to the ATCC guidelines . In short-test was employed to compare two treatment groups. A p-value of <0.05 was considered statistically significant.All experiments were performed at least in 3 independent replicates, and the results are presented as mean ± standard error (SE). Data points were analyzed by GraphPad Prism software . A one-way analysis of variance (ANOVA) and the Bonferroni post hoc test were used to analyze multi-treatment groups, whereas Student’s K. pneumoniae bacteria. This study concluded that four out of the six tested AgNP-based combinations  had synergistic activities against all tested Gram-negative bacteria (except vancomycin in AMR strain I). One important finding is that the MIC values of both wild-type and AMR bacteria were similar following exposure to AgNPs, hence suggesting that AgNPs act via a mechanism or multiple mechanisms through which the tested bacteria remain susceptible to them. However, further studies are required to confirm this hypothesis. Finally, exposing the AgNP-based combinations at the synergistic concentrations to mammalian cells, namely HepG2, demonstrated minimal toxicity, particularly at concentrations < 16 µg/mL.AMR bacteria have emerged as a global health threat, and currently, there is an overall shortage in the development of new antimicrobials. In recent years, AgNPs have shown promising antimicrobial properties; however, the relevant dosage of AgNPs in combination with different antimicrobials is rarely known. Additionally, the cytotoxicity of AgNPs within synergistic combinations to mammalian cells is seldom reported. Therefore, this study sought to investigate AgNP-based combinations with different classes of antimicrobials against wild-type Gram-positive and -negative bacterial strains as well as clinically isolated AMR K. pneumoniae, since emerging evidence is demonstrating the increasing prevalence of local infections caused by AMR K. pneumoniae [Although the previous reports discussed earlier demonstrated great antimicrobial potential for AgNPs against different types of bacteria, including AMR bacteria, there remain inconsistencies with regard to the used strains, the physicochemical properties of AgNPs, the dosing and antimicrobial combinations, and the cytotoxicity in mammalian cells ,37,38,39eumoniae . Furthereumoniae ,67,73,92


Klebsiella aerogenes in Brazil. Eight polymyxin-resistant and carbapenemase-producing K. aerogenes strains were isolated from patients admitted to the ICU of a tertiary hospital. Bacterial species were identified by automated systems and antimicrobial susceptibility profile was confirmed using broth microdilution. The strains displayed a multidrug resistant profile and were subjected to whole-genome sequencing. Bioinformatic analysis revealed a variety of antimicrobial resistance genes, including the blaKPC-2. No plasmid-mediated colistin resistance gene was identified. Nonetheless, nonsynonymous mutations in mgrB, pmrA, pmrB, and eptA were detected, justifying the colistin resistance phenotype. Virulence genes encoding yersiniabactin, colibactin, and aerobactin were also found, associated with ICEKp4 and ICEKp10, and might be related to the high mortality observed among the patients. In fact, this is the first time ICEKp is identified in K. aerogenes in Brazil. Phylogenetic analysis grouped the strains into two clonal groups, belonging to ST93 and ST16. In summary, the co-existence of antimicrobial resistance and virulence factors is deeply worrying, as it could lead to the emergence of untreatable invasive infections. All these factors reinforce the need for surveillance programs to monitor the evolution and dissemination of multidrug resistant and virulent strains among critically ill patients.This study evaluated the scope and genetic basis of polymyxin-resistant  Klebsiella aerogenes represents one of the bacterial species causing nosocomial infections; it also causes respiratory infections, urinary tract infections, endocarditis, skin or soft-tissue infections, abdominal infections, and osteomyelitis [myelitis ,2,3. Mormyelitis ,5.Enterobacteriaceae is an urgent threat to human health because polymyxin is regarded as a drug of last resort in the therapeutic management of Gram-negative infections that otherwise have limited or no therapeutic options [Klebsiella spp. may harbor several virulence factors associated with survival and pathogenesis, including a e integrative conjugative element (ICEs), which is a group of mobile genetic element transferable between bacterial species, associated with hypervirulence in K. pneumoniae strains  [Polymyxin-resistant  options . Resista options . In addi options . Furtheribactin) ,10,11,12Enterobacter spp. have been limited compared with those on other Enterobacteriaceae pathogens. In this study, we identified the genetic mechanisms associated with polymyxin resistance in K. aerogenes isolates from a tertiary hospital located in the Central-Western region of Brazil by using whole-genome sequencing.Effective treatment of infections caused by these pathogens as well as the implementation of adequate preventive measures to effectively contain the spread remain challenging . VariousEnterobacteriaceae infection were identified. Twenty-five strains were isolated from patients presented to the adult intensive care unit (ICU). Of these, polymyxin-resistant and carbapenemase-producing K. aerogenes strains were isolated from eight patients. The majority of patients were female (62.5%), with a median age of 45 years . No significant differences (p > 0.05) in baseline demographics were observed among the patients. All patients had a diagnosis of infectious diseases at the sample collection time. Four of them had bloodstream infections, whereas the remaining patients had a diagnosis of pulmonary and urinary infections. All patients were exposed to antibiotics 30 days prior to the initial detection of the polymyxin-resistant strain , belonging to clonal complex 3 (CC3), and ST16 (n = 3). ST93 has been previously described in clinical isolates of K. aerogenes in Brazil [K. aerogenes clones associated with human infections [https://pubmlst.org/bigsdb?db=pubmlst_kaerogenes_isolates&page=query, accessed on 18 May 2022). The high genetic similarity of strains suggested transfer events between patients. Therefore, measures must be implemented to avoid nosocomial transmission [Regarding phylogenetic analysis and virulome, the core genome phylogeny and MLST analysis identified similar populations and were used to classify the strains into two strongly clonal groups. On the basis of SNPs, a maximum-likelihood phylogenetic tree was constructed, demonstrating that strains were closely related and partitioned into two clades. MLST analysis identified two different sequence types: ST93  lineages  were found associated with two different structural variants of ICEKp (ICEKp4 and ICEKp10)  . ICEKp ipulation . To the  ICEKp10 . In factvariants . As ICEK50 ranging from 8 mg/L to 32 mg/L). Resistance gene profiles varied among strains, with some harboring several beta-lactam, aminoglycoside, tetracycline, quinolones, sulfonamides, trimethoprim, amphenicol, fosfomycin, macrolide and rifamycin resistance determinants. The blaKPC-2 and blaOXA−1 carbapenemase genes were identified in all isolates. In addition, the strains carried the blaCTX−M−15 , blaTEM-1B  and blaOXA−9 groups  extended-spectrum β-lactamase (ESBL) encoding genes. Although it was not possible to determine the location of these antimicrobial resistance genes, six different plasmid replicons were identified among these isolates (Regarding the susceptibility profile  Table A2isolates . This fimcr-like) were not detected. Nonetheless, deleterious point mutations were found in mgrB, pmrA, pmrB, eptA, and arnT genes . A second potential mechanism of mutational colistin resistance, due to a substitution at amino acid position (T296S) in eptA, was identified in all ST93 isolates (62.5%). A third potential mechanism of mutational resistance to colistin, due to an amino acid position substitution in pmrAB, was observed in the ST16 isolates (37.5%).Plasmid-encoded colistin resistance genes  according to the manufacturer’s instructions. After isolation, the susceptibility profile was confirmed and minimal inhibitory concentrations (MICs) of antimicrobials were determined using broth microdilution, following the Clinical and Laboratory Standards Institute guidelines [Identification and screening of antimicrobial-resistant bacterial species were performed using Phoenixidelines . The folidelines .® DNA Mini Kit . The concentration and purity of DNA were determined using a Qubit® 2.0 fluorometer and the dsDNA BR Assay Kit . Sequencing libraries were prepared using the Nextera library kit . The prepared libraries were sequenced with 150 bp paired-end reads via IlluminaMiSeq Platform , as described in a previous study [K. aerogenes core genome was defined as the concatenation of coding sequences presenting one copy in all final assemblies [Genomic DNA was extracted from fresh cultures by using QIAampus study . FastQC us study . Each reus study  with k-mus study . Speciesus study . The K. semblies . The whoKlebsiella aerogenes ATCC 13048). Maximum likelihood phylogenetic trees were constructed using RAxML 8.1.23 [K. aerogenes MLST database [Single nucleotide polymorphisms (SNPs) were identified through mapping of Illumina reads to a reference genome  for prediction of acquired antimicrobial resistance genes. Chromosomal genes mgrB, phoP, phoQ, pmrA, pmrB, eptA, and arnT were manually screened for point mutations associated with colistin resistance by using blastn and blastx tools, and Klebsiella aerogenes ATCC 13048 was used as reference genome (GenBank accession number QVMZ00000000.1). PROVEAN v. 1.1.3 software was used to predict the functional effect of each found mutation, considering deleterious mutations as related to the resistant phenotype [http://www.mgc.ac.cn/VFs/main.htm, accessed on 6 February 2022) and ICEKp variants were determined by using blastx. A ≥98% threshold for sequence identity was used for resistance and virulence genes identification (Assembled genomes were submitted to ResFinder 4.1 (henotype . Plasmidhenotype . Virulenfication .mgrB was the primary reason behind polymyxin-resistance development. To the best of our knowledge, so far, this is the first report identifying ICEKp in isolates of K. aerogenes in Brazil, namely ICEKp4 and ICEKp10. A careful and continued surveillance system providing epidemiological and molecular information is required to follow the evolution of polymyxin resistance in Brazil and to limit the risk of outbreaks caused by these high-risk clones.Our findings demonstrated that chromosomal alteration in


Background:Pseudomonas aeruginosa outbreaks can originate from various sources and can cause severe complications in posttransplant patients. Antibiotic prophylaxis can decrease posttransplant infections; however, consideration must be given to P. aeruginosa coverage as we outline an outbreak among the stem-cell transplant (SCT) population. Methods: A multidisciplinary outbreak investigation was conducted to evaluate sources of contamination and changes in clinical processes. Positive blood cultures from SCT patients and environmental isolates were analyzed using whole-genome sequencing (WGS). Incidence density rates for P. aeruginosa blood cultures from January 2019 through October 2020 were calculated per 10,000 patient days and stratified by unit, specimen, and transplant type. Statistical analysis was calculated with significance at p < 0.05. Results: A cluster of 8 SCT patients was identified between May and September 2020. Moreover, 10 environmental samples were positive for P. aeruginosa including drains, water sources prior to the point-of-use (POU) filter and blood-bank thaw machines. Phylogenetic analysis revealed 1 cluster of 2 patients who shared the same room, 5 patients with unique P. aeruginosa isolates, and 2 separate clusters of environmental isolates with relatedness only to each other. Review of clinical processes showed a change from fluoroquinolone prophylaxis to cephalosporin in the spring of 2020. Also, 5 P. aeruginosa bacteremia infections occurred prior to June . During the period of cephalosporin use, 8 infections were identified  (P = .006). Following the restart of fluoroquinolone, zero infections have occurred to date, as of January 28, 2021. Conclusions: Discontinuation of fluoroquinolone prophylaxis was associated with P. aeruginosa bacteremia infections in SCT patients. Use of fluoroquinolone prophylaxis in SCT patients is protective from P. aeruginosa bacteremia infections. There have been no further infections in the following 3 months after the change back to the use of fluoroquinolone. Additionally, WGS showed that most patient isolates did not have a common source, suggesting that P. aeruginosa gastrointestinal colonization may play a role in seeding these bacteremia infections.Funding: NoDisclosures: None


Klebsiella variicola is a pathogen that is increasingly recognized as being associated with human infections, but the methods available to clinical microbiology laboratories for accurate identification are limited. In this study, we assessed the accuracy of identification of K. variicola by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry using genetic identification with multiplex PCR as the reference method. Antimicrobial susceptibilities and virulence of K. variicola strains were also investigated. Fifty-five Klebsiella pneumoniae, 26 K. variicola, and 2 Klebsiella quasipneumoniae clinical strains were used for evaluation. Both MALDI Biotyper with library version 9 and Klebsiella MALDI TypeR, a web-based species identification tool using MALDI-TOF data, accurately identified all K. variicola strains. In addition, two strains of K. quasipneumoniae were accurately identified with Klebsiella MALDI TypeR. Whole-genome sequencing confirmed the accurate identification to the subspecies level by Klebsiella MALDI TypeR for four strains (two strains each of K. variicola subsp. variicola and K. quasipneumoniae subsp. similipneumoniae). While 13 strains, 3 strains, and 1 strain of K. pneumoniae showed nonsusceptibility to ampicillin-sulbactam, ceftriaxone, and meropenem, respectively, all strains of K. variicola were susceptible to all tested antimicrobial agents. Although two K. variicola strains were positive for the string test, no K. variicola strains harbored any of the genes associated with hypervirulence of K. pneumoniae. Accurate identification of the K. pneumoniae complex, including K. variicola, by MALDI-TOF in clinical microbiology laboratories is expected to clarify the clinical characteristics of each species in the future.IMPORTANCE Recent widespread use of bacterial whole-genome sequencing analysis has resulted in the proposal of novel bacterial species and reclassification of taxonomy. Accurate methods for identification of bacterial species in clinical microbiology laboratories are essential to accumulate information on the clinical characteristics of each bacterial species. Klebsiella variicola is a member of the Klebsiella pneumoniae complex, and its association with human infections has been increasingly recognized, but accurate identification methods approved for use in clinical microbiology laboratories have been limited thus far. The findings of the present study suggest that K. variicola can be accurately identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry using updated library or web-based identification tools. Accurate identification will promote exploration of clinical characteristics of K. variicola. Klebsiella variicola was first reported as a plant-associated bacterium; it was later shown to cause human infections  gene is the most reliable method  and K. variicola (n = 16) recovered from blood culture and identified with a score of 2.00 or higher by MALDI Biotyper  at the University of Tokyo Hospital (UTH) from January 2019 to July 2020. After the microbiology laboratory of UTH updated the library from version 4 to 9 in August 2020, 15 strains recovered from blood culture were identified as K. variicola with a score of 2.00 or higher until November 2021, and these strains were added for the analysis. Only one strain from each patient was used. K. pneumoniae FUJ00227 (accession no. JAMOBN000000000), Klebsiella quasipneumoniae subsp. similipneumoniae FUJ00228 (accession no. JAMOBM000000000), and K. variicola subsp. variicola FUJ01370 (accession no. GCA_019042755), bacterial species of which have previously been identified by whole-genome sequencing and >99% average nucleotide identity (ANI) with type strains using ANI Calculator (https://www.ezbiocloud.net/tools/ani), were added as reference strains  using MALDI-TOF data obtained by MALDI Biotyper , which were reported as biomarkers for hypervirulent K. pneumoniae . The MICs were evaluated using CLSI M100-S27 . Althougl agents . Virulence genes . While teumoniae .K. variicola strains with hypermucoviscosity was performed with Illumina MiSeq  as described previously (https://pathogen.watch/). Species identified by whole-genome sequencing were identical to those identified by multiplex PCR  (Whole-genome sequencing of (i) strains with discordant bacterial identification results by MALDI-TOF and multiplex PCR and (ii) eviously . Raw reaplex PCR . Accuratand iuc) .K. pneumoniae complex with MALDI-TOF by identifying the characteristic peak position  that confers hypermucoviscosity to K. variicola in an rmpA-independent manner has been reported, plasmid analysis is beyond the scope of our study (Fatal infections and multidrug-resistant strains of reported , 17. Ourclusions , 19. It fections . While tur study .K. variicola (Recent studies have revealed the molecular characteristics of ariicola , 21. AccPRJNA858671.All genome sequences have been deposited in the NCBI database under BioProject accession no.


Astragalus candolleanus (A. candolleanus) hydromethanolic root extract against Gram-positive (Bacillus subtilis and Staphylococcus aureus) and Gram-negative  strains by the cup-plate method. The root was powdered and extracted with 70% methanol by cold maceration for 5 days. Preliminary phytochemical screening was performed with different solvents in the order of increasing polarity. Pure compounds were isolated by column chromatography and were characterized through liquid chromatography-mass spectrometry. Targeted predictions of the isolated compounds were also studied using Swiss Target prediction software and prediction of activity spectra for substances. The extract showed a broad zone of inhibition against pathogenic bacteria. Four pure compounds were isolated, of which a novel terpenoid compound has been identified as stemmadenine along with scillirosidin, cephalotaxine, and myxoxanthophyll. The structures of the isolated phytoconstituents were elucidated by spectral analysis. The four pure components isolated from the roots of A. candolleanus are suggested to be effective against tested pathogens. Overall results of drug design suggest that myxoxanthophyll is a promising bioactive compound endowed with antibacterial activity.Antibiotic resistance represents one of the biggest challenges, and there is an urgent need for plant-based antimicrobial agents that enable managing this crisis effectively. In this work, we aimed to investigate the antibacterial activity of  Astragalus rhizanthus subsp. Candolleanus Benth. (synonym Rudravanti) (A. candolleanus) belong to the family Fabaceae, and it is a wild-growing herb widely spread in the Himalayas from Jammu & Kashmir to Uttarakhand provinces in India [A. candolleanus is endowed with several health benefits, such as immune-boosting, antiaging, and anti-inflammatory effects [Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Bacillus subtilis (B. subtilis), Pseudomonas aeruginosa (P. aeruginosa), Klebsiella pneumoniae (K. pneumoniae), and Kocuria rhizophila (K. rhizophila) [Astragalus angulosus ethanolic extract against three Gram-positive strains  and two Gram-negative strains (E. coli and P. aeruginosa) [Astragalus species  endemic to Turkish flora. Specifically, they performed the disk diffusion assay against P. aeruginosa (low activity with respect to standard antibiotics tetracycline and oxacillin) and Candida albicans (no activity) [Astragalus membranaceus ethyl acetate aerial parts extract. In particular, it was effective against the Gram-positive strain B. subtilis [A. candolleanus root extracts. Considering that literature data reported outstanding biological activity of the root extracts from other Astragalus species, we proposed to fill the knowledge gap on A. candolleanus by investigating the chemical profile and antimicrobial activity of its extract. After a thorough literature survey on Astragalus species with emphasis on its phytochemical screening in various parts of the plant , the root extract was considerably found to be enriched with phytoconstituents [Astragalus mongholicus is colonized by several bacterial species, which are able to modify the secondary metabolites of the plant. According to such a study, the biological properties and chemical features of a phytoextract can be the results of the interaction between bacteria and plants [A. candolleanus root extract against the most common bacteria involved in septicemia and, subsequently, identify the active phytoconstituents responsible for the biological activity. For this purpose, by column chromatography, the root extract was fractionated to isolate the various pure phytoconstituents. These pure components were elucidated using the mass spectrometry technique.Antibiotic resistance represents a serious problem for public health , 2. Despin India , 9. A. c effects , 11. Thi effects –15. A fezophila) . Interesuginosa) –19. Albactivity) . Recentlsubtilis . This acsubtilis . Howevertituents –26. Inted plants . So, theAll the reagents were of analytical grade and purchased from Merck & Hi Chem Life Sciences except those mentioned elsewhere.A. candolleanus were collected from Losar  district of Himachal Pradesh. The plant's identity was confirmed by an acknowledged botanist, Dr. Sunita Garg, Emeritus Scientist, CSIR-NISCAIR Raw Material Herbarium and Museum, New Delhi (RHMD). A specimen voucher (NISCAIR/RHMD/Consult/2018/3253–54) was deposited at NISCAIR.The roots of A. candolleanus roots were dried at room temperature and reduced to a coarse powder. About 20 g of the powdered root was extracted with 200 mL of methanol-water mixture (7 : 3 v/v) by cold maceration for 5 days. Afterwards, the mixture was decanted and filtered to get the crude extract. The extract was then concentrated under reduced pressure through a rotavapor (Buchi-R100), followed by drying on a desiccator.n-hexane, petroleum ether, chloroform, methylene chloride, acetone, ethyl acetate, methanol, ethanol, water, methanol : water (7 : 3 v/v), and n-butanol : acetic acid : water (BAW) (4 : 1 : 5 v/v). The powdered root extract along with different solvent systems was allowed to stand for 48 hours. After filtration, chemical tests allow the qualitative analysis of the extract [One milligram of the powdered roots was macerated individually in volumetric flasks containing different solvents, including dimethyl sulfoxide (DMSO),  extract , 28. TheA. candolleanus was evaluated by the cup-plate method against B. subtilis (ATCC 6633), E. coli (ATCC 9637), K. pneumoniae (ATCC 10031), K. rhizophila (ATCC 9341) P. aeruginosa (ATCC 25619), Salmonella typhimurium (ATCC 1428), and S. aureus (ATCC 6538). Bacteria were cultured on nutrient agar media (Hi-media). The method of Ali et al. with some modifications was used for the antibacterial assay [μL of extract (100 mg/mL) in different dilutions (from 5 to 80 μg/ml) was placed in the wells along with DMSO as a negative control. DMSO was diluted in a 1 : 100 ratio and did not affect bacterial growth. The activity of the natural extract was compared to that of the standard antibiotic gentamycin, in concentrations ranging from 5 to 40 µg/ml. The agar plates were incubated for 24 h at 30–35°C. The parameters used for observation were the estimation of the inhibition zone of bacterial growth surrounding the wells. The unit for the diameter of the inhibition zone was taken in millimetres (mm).The bacterial strains were purchased from the American Type Culture Collection Centre (ATCC) through an authorized vendor, Hi-Media Pvt. Ltd. Resources (reagents and apparatus) from the Indian Pharmacopoeia Commission, Ghaziabad, and used to carry out this research. The antibacterial activity of hydro-methanolic root extract of  ATCC 256, Salmonev/v ethanol-water. The isolated and purified compounds were analyzed using Fourier-transform infrared spectroscopy  and liquid chromatography-tandem mass spectrometry (LC-MS-MS) (Agilent 6520).A column of 400 mm length with 500 mL reservoir capacity, 30 mm internal diameter, and 40 mm outer diameter was prepared with the wet packing method using silica (100–200 mesh size) as a stationary phase. The hydro-methanolic root extract was packed into the column and was eluted in a sequence from nonpolar solvents to polar solvents to obtain different pure fractions. The similarity profile of fractions was checked by thin layer chromatography, and similar fractions were identified. Pure components could be obtained in the case of root extract eluted with 70% The melting point is an intensive physical property that is characteristic of a specific compound. Thus, the melting points of the isolated compounds were determined to ensure their purity. All melting points were measured on a melting point apparatus .https://www.swisstargetprediction.ch/) [https://www.way2drug.com/) [The isolated compounds were subjected to Swiss Target Prediction (STP) (ion.ch/)  and Predug.com/) –31 to unA. candolleanus showed the presence of alkaloids, glycosides, terpenes, lipids, volatile oil, gums, and mucilage. After evaporation of the solvent, the screening for active phytoconstituents in the semisolid hydro-methanolic root extract of A. candolleanus was able to inhibit the growth of all tested Gram-positive and Gram-negative strains. Specifically, as reported in µg/mL; however, at the concentration of 10 µg/mL, it produced a broader inhibition zone against S. aureus (28.6 mm), S. typhimurium (25.5 mm), K. rhizophila (24.6 mm), K. pneumoniae (21.8 mm), and E. coli (22.7 mm). Besides, at this concentration, A. candolleanus extract showed a strong growth inhibition for B. subtilis (41.2 mm) and P. aeruginosa (41.6 mm). These bacteria are seen to be mainly responsible for recurrent bacterial infections. The obtained results indicated that the hydro-methanolic root extract of A. candolleanus can be considered a promising antimicrobial agent, due to its broad zone of inhibition against pathogenic bacteria. Our results revealed that the extract was more efficient in counteracting the growth of Gram-positive strains compared to the Gram-negative ones, except for P. aeruginosa. In this regard, it is worth noting that Gram-negative bacteria are generally more resistant to the natural antimicrobial agents compared to the Gram-positive ones. This condition reflects the different composition of the cell wall between the two types of bacteria [A. candolleanus allows the identification of pure compounds. The isolated and purified compounds were analyzed using FTIR and LC-MS-MS techniques. The results and the inference of the characterization of A. candolleanus root by FTIR are presented in A. candolleanus root extract is shown in The hydro-methanolic root extract of bacteria . The bacbacteria , 34. Thibacteria . Concernbacteria . The chaA. candolleanus have been identified. Four compounds have been isolated and identified using the data of peaks of mass spectral analysis . The compound identified from peak 3 is an alkaloid—cephalotaxine . The compound identified from peak 4 is a terpenoid—stemmadenine . The compound identified from peak 5 is a myxol glycoside—myxoxanthophyll .Results of LC-MS-MS of A. candolleanus, further studies should be conducted on these microorganisms to confirm their effectiveness as well as to elucidate the mechanism of action through which they exert the antibacterial activity. In response to bacterial infection, high levels of proinflammatory cytokines, such as interleukin-6 (IL-6), IL-8, IL-18, tumor necrosis factor-alpha (TNF-α), and anti-inflammatory cytokine (IL-10) were often found in infected patients. A decrease in IL-6 was associated with a better prognosis instead, and overproduction of IL-10 is considered the main predictor of severity and fatal outcome. In bacterial infections, proinflammatory and anti-inflammatory cytokines are a double-edged sword: on the one hand, they are essential for eradicating the pathogen, but their overproduction can cause tissue and organ damage [A. candolleanus, identified through the STP software. Out of the four compounds tested, we found that scillirosidin and stemmadenine inhibited MAP kinase p38 alpha and beta pathways, responsible for an inflammatory imbalance during bacterial infections, whereas myxoxanthophyll and scillirosidin also target interleukin-8-receptor A and B, which are the major proinflammatory cytokines that get elevated in such patients. The PASS online predictivity scores for bioactive compounds isolated from the roots of A. candolleanus are reported in According to earlier studies, terpenoids are found to be more effective against Gram-positive bacteria than Gram-negative bacteria due to their lipophilic properties. Monoterpenes preferentially affect the membrane structures by enhancing the permeability as well as changing the structural arrangement of proteins, producing interference inside the respiratory chain . The natn damage , 45. TheA. candolleanus extract was tested against several microbial strains, including B. subtilis, S. aureus, E. coli, S. typhimurium, P. aeruginosa, K. pneumoniae, and K. rhizophila, and results showed a broad inhibition zone against bacterial species. The activity may be a cumulative effect of all the constituents present in the plant. In our study, we have isolated four compounds that were identified as scillirosidin, cephalotaxine, stemmadenine, and myxoxanthophyll using FTIR and LC-MS-MS techniques. These compounds will be further tested individually against bacterial strains. We also performed computational studies to predict the most active constituent amongst all four compounds. The results of STP as well as PASS online predictivity score software testing showed that, among the four compounds, myxoxanthophyll was the most active molecule, revealing a predicted activity as an apoptosis agonist, antioxidant, and anti-inflammatory compound, with a Pa score above 0.7. However, this study can be considered a preliminary investigation of the chemical composition and biological activities of A. candolleanus. Further studies are required to validate the activity of these identified compounds using antimicrobial and docking studies.The antibacterial activity of