The ts3agn mutation drastically increases the frequency ofectopic contacts (FEC) in specific regions of intercalary heterochromatin, suppresses learning/memory and affectslocomotion. As is shown in this study, the polytene X chromosome bands in reciprocal hybrids between ts3agn and thewild type strain Berlin are heterogeneous in modes of FEC regulation depending either on maternal or paternal geneorigin. Bioinformatic analysis reveals that FEC between X:11AB and the other X chromosome bands correlates with theoccurrence of short (~30 bp) identical DNA fragments partly homologous to Drosophila 372-bp satellite DNA repeat.Although learning acquisition in a conditioned courtship suppression paradigm is similar in hybrids, the middle-termmemory formation shows patroclinic inheritance. Seemingly, this depends on changes in miR-974 expression. Severalparameters of locomotion demonstrate heterosis. Our data indicate that the ts3agn locus is capable of trans-regulatinggene activity via POEs on the chromatin nuclear organization, thereby affecting behavior.Prognosis of neuropsychiatric disorders in progeny requires consideration of individual (1) parent-of-origineffects (POEs) relying on (2) the nerve cell nuclear 3D chromatin architecture and (3) impact of parent-specific miRNAs.Additionally, the shaping of cognitive phenotypes in parents depends on both learning acquisition and forgetting,or memory erasure. These processes are independent and controlled by different signal cascades: the first is cAMPdependent,the second relies on actin remodeling by small GTPase Rac1 – LIMK1 (LIM-kinase 1). Simple experimentalmodel systems such as Drosophila help probe the causes and consequences leading to human neurocognitive pathologies.Recently, we have developed a Drosophila model for Williams–Beuren Syndrome (WBS): a mutant  Genome plasticity is ensured by the architecture of specificnuclear loci and nuclear localization of transcriptional machinerywhere the chromatin organization is the priority-driven factor. The outcome of recent achievements in systems biologyis the notion that the plasticity of 3D chromatin architectureof nervous cell nuclei plays the leading role in cognitionand neuropsychiatric disorders . The epigeneticcomponent is still an underestimated source of psychomotordisturbances and neuronal diversity . Therefore, a new field of human biomedical researchnamed molecular cytogenetics and cytogenomics , or chromosomics  has evolved. Themain goal of chromosomics is the study of chromosomes,their 3D architecture in the interphase nucleus, the outcomesof chromosomal sub-region plasticity and gene interactions forshaping interindividual and intercellular genomic variationsin normal behavior and disease.Recently, this topical problem has turned out to be thepursuit of understanding the concomitant role of activeforgetting, since the antithesis to learning acquisition is theforgetting or memory erasure . Bothprocesses are independent and controlled by different signalcascades: learning acquisition and memory consolidationoccurs via cAMP cascade, its components being CREB andC/EBP. Active forgetting relies on actin remodeling cascaderesponsible for structural alterations of neurons and synapses:small GTPase Rac1 – LIMK1 (the key enzyme of actin remodelingLIM-kinase 1) and its phosphorylation substrateсofilin. The absence of Rac1-dependent forgetting causesthe autistic spectrum disorders. Expression changes (activeor non-active state) of LIMK1 and cofilin lead to differentneuro pathologies. The most studied example embracing allthe aforementioned facets of manifestations is Microdeletion(Deletion) Williams–Beuren Syndrome, or WBS in 7q11.23.WBS deletion leads to cardiovascular pathology, cognitivedeficit in visuospatial construction and hypersociability. This isbecause long-term synaptic plasticity and depression determinesuccessfulness of learning and memory, as well as oflocomotor behavior, and depend on epigenetic regulation of LIM-kinase 1 (LIMK1) gene, one of approximately 28 genesuncovered by WBS deletion. Epigenetic regulation of LIMK1activity involves DNA methylation, chromatin remodeling andthe noncoding RNA-mediated process .LIMK1, a member of serine/threonine (Ser/Thr) familykinases regulated by the Rho-GTPase pathway, is the keyenzyme of actin remodeling cascade. Dendritic spines areactin-rich structures, and spine dynamics is driven mainly byactin remodeling, thus sharing several molecular pathwayswith dendrite growth . Increasing sets ofevidence suggest that nuclear actin also plays a pivotal rolein transcriptional regulation and DNA repair. Interestingly,monomeric actin is a stoichiometric subunit of a variety ofchromatin remodeling complexes. Ashift between monomericand polymeric states modifies activity of histone deacetylases.agnostic locus harboringLIMK1 gene (X:11AB). This region possesses theproperties of intercalary heterochromatin. Being a hotspot ofchromosome breaks, ectopic contacts, underreplication andrecombination, the region attains strain-specific architecturemarked by single base changes and small insertion/deletions.The EMS-induced temperature-sensitive (ts) mutationts3agn carries the insertion of transposable element (TE) fromTc1/mariner superfamily ~460 bp downstream 3′UTR ofDrosophila LIMK1 gene (dlimk1), as well as A/T-rich 28 bpinsertion within intron 1 of dlimk1 capable of pairing with5′ TIR of the TE.Recently, we have developed a simple and appropriateDrosophila model for chromosomics  using the properties of the ts3agn shows a temperaturesensitivelethality at all stages of development except for theimaginal stage. At normal temperature, the adult flies showdrastic learning acquisition and memory retention defects, aswell as locomotor impairments and amyloid-like inclusions. Stressexposure (heat shock for 30 min at 37 °C) suppresses thesemanifestations . Also, ts3agnmutationleads to: (1) LIMK1 and p-cofilin increase in the adultbrain and salivary glands of 3rd instar larvae at 22–25 °C anda fall down to the level of the wild type strain Canton S at29–37 °C; (2) high level of ts-induced recombination withints3agn region; (3) 3-fold increase in frequency of non-allelicectopic contacts (FEC) within 2L arm of the chromosome 2 and in the 11В X chromosome region . Additionally, miRNAs expression including the biomarkersfor human neuropathologies is drastically reducedin ts3agn relative to the wild type strains .When maintained at 29 °C, ts3agn-specific nuclear organization is shaped in early embryogenesisalongside with formation of chromosomal heterochromatinregions. Intrinsic ts3agn FEC is maternally inherited. Therefore, ts3agn is a promisingmodel for studies on parent-of-origin effects (POEs) on progenyconsidered as significant causative factors of psychiatricdisorders . For instance, a 1.5 Mb WBSdeletion recurrently arises de novo and depends on POEs: maternalorigin leads to more severe developmental abnormalitiesand microcephaly . Moreover,when WBS deletion has a paternal origin, expression levels ofa number of genes within the WBS deletion decrease. Amongthese genes crucial for the brain development is a gene for generaltranscription factor II-I (GTF2I). It regulates transcriptionby binding to DNA and histone deacetylase (HDAC) . The main goal of the study is the analysisof POEs role in shaping quantitative traits, namely learningacquisition, memory retention, locomotion, and miRNAs expressionwhile using the advantages of the Drosophila modelfor POEs in progeny from reciprocal crosses between ts3agnand the wild type strain Berlin .To meet the requirements of chromosomics, FECs betweenthe region X:11AB and the other bands of the X chromosomemay be estimated as an indicator of chromosomal spatialorganization. This approach is justified by the existence oflate-replicating genomic territories including the underreplicatedregions of polytene chromosomes. These regions overlapwith late-replicating regions of mitotically dividing cells. Suppression of SUUR gene responsiblefor underreplication of intercalary heterochromatin and, as aconsequence, for the ectopic pairing leads to death in early embryogenesis. Additionally, to elucidatethe contribution of DNA sequence homology as componentsof epigenetic regulation in the ectopic chromatin pairing wehave developed the special software package Homology SegmentAnalysis.Drosophila stocks. The fly stocks used belong to Biocollectionof Pavlov Institute of Physiology of the Russian Academyof Sciences:• Berlin, a wild type strain;• Canton S, a wild type strain;• ts3agn, a temperature-sensitive mutation on Canton Sgenetic background within agnostic locus (X:11AB) affectingdlimk1 activity.ts3agn and Berlin were usedbecause Berlindlimk1 sequence is closer to FlyBase referencesequence . At the same time,the reciprocal hybrids ts3agn×Canton S (the genetic backgroundfor ts3agn) and Canton S×ts3agn demonstrate exactlythe same cognitive behavior as reciprocal hybrids with Berlin approving the usage of Berlin.The reciprocal hybrids between Berlin and ts3agn female and male parents and F1 female and male progeny from reciprocal crosses with theaccent on the putative hairpin formed in the X chromosomeby 28 bp A/T rich insertion within intron 1 of dlimk1 geneand 3′ end of Tc1/mariner element.Figure 1 shows the X chromosome and autosomes architecturein Flies were maintained on standard Drosophila yeast-raisinmedium at +22 ± 0.5 °С under a 12-h light/dark cycle. Forthe memory and locomotion tests, males were collected uponeclosion without narcotization and kept individually in culturevials till the behavioral experiments on the 5th dayEstimation of frequency of ectopic contacts (FECs).The aceto-orcein squash preparations were prepared fromsalivary glands of III instar D. melanogaster female larvae.20 to 30 animals were examined, therefore the number ofanalyzed chromosomes varied from 300 to 500. The examplesof ectopic contacts are presented on Fig. 2. The number ofnon-homologous contacts between the region X:11AB anddifferent bands of the X chromosome was calculated and expressedas per cent of the total number of the examined nuclei.FECs in parents and F1 reciprocal hybrids were comparedusing Student’s t-test. Identification of genes localized in theX chromosome bands forming contacts with the 11AB regionwas performed using NCBI Genome Data Viewer databasehttps://www.ncbi.nlm.nih.gov/genome/gdv/ and molecularfunction of identified genes was derived from FlyBase https://flybase.org analysis of DNA segments homology.D. melanogaster genome sequence (release 6) was taken from. Special software package HomologySegment Analysis searching the matches of short singlestrandedDNA fragments within the chromosome areasinvolved in the ectopic pairing in Drosophila has been developed.The software written in Python 3 can be freely downloadedfrom . Software version from git(commit 41719cddc6283edbd79c5bf2aee237cde48d4b7d)was used. The algorithm of the program is described in brief in. The exact run parameters for 11AB region are following: segmentanalysis.py -v -s 30 dm6.nounmapped.fa.gz :X:11982050:12772070 DmelMapTable.160615c.bedHere, dm6.nounmapped.fa.gz is Drosophila genome canonicalsequence (Ensembl release 6) and DmelMapTable.160615c.bed is chromosome bands location from Ensembldatabase converted to BED format. Both files are included insoftware repository.For other tested regions, all parameters were the sameexcept for the regions location.Preparation of miRNAs libraries and bioinformaticanalysis. The detailed description of the procedure is givenin . Extract RNA reagent was used for total RNA extraction fromadult 5 days old males. To obtain the fraction of small RNA,25 μg of total RNA were separated using 15 % polyacrylamidegel electrophoresis in the presence of Urea (8 M) followingexcision of small RNA fraction corresponding to 21–29 nts.Illumina TruSeq Small RNA prep kit  wasused for small RNA libraries preparation. Sequencing wasperformed on an Illumina HiSeq 2000 platform.The amount of mapped miRNAs reads was counted byBEDTools (v. 2.22) and mirbase annotation (r. 19) . Analysis of differentially expressed miRNAs wasperformed using edgeR (v. 3.10.2) package in R environment(v. 3.2.2) . miRNA functions werederived from miRBase . Small RNAlibraries were deposited in NCBI SRA under the numberPRJNA633483.Locomotor activity. Computer-aided automatic device forsimultaneous registration of 20 animals is described in . The experiment lasted for 1 h. Spontaneouslocomotor activity of flies was detected in a plate witheight chambers and transparent cover using high-resolutionvideo camera. Software used for locomotor activity analysisis freely available at .The following parameters of locomotion were assessed:activity index (%); run frequency (the number of run boutsin 100 seconds); running speed (mm/s). The full record wasdivided into 1 s quanta, and the mean speed of fly movementin each quantum was calculated. If the result was less thanthe threshold value (5 mm/s), the fly was considered to beresting during this time quantum; otherwise, it was considered moving. Neighboring quanta with similar movement patternwere merged in intervals of moving and resting. Activity indexis determined as a time spent in movement. Running speedis an average fly speed, determined using only intervals ofmovement. The Kruskal–Wallis analysis of variance with themultiple comparison of mean ranks was used to compare allthe experimental groups.Learning acquisition and middle-term memory formationin Drosophila males. Detailed description of learning/memory assessments in conditioned courtship suppressionparadigm (CCSP) and specially designed software for observationand statistical analysis (randomization test) of learningindices based on courtship indices is given in . CCSP employs the natural stimuli of Drosophilacourtship. Both virgin and fertilized females emit an aphrodisiacpheromone, attracting a naïve male without courtshipexperience. However, a fertilized female rejects a male atthe courtship stage of attempted copulation via emitting anaversive pheromone. Repetitive rejections during 30 mintraining provoke a kind of learned helplessness when a malestops courting another female. This courtship suppressionmight last for one hour when test female is virgin and foreight hours when fertilized. Males with defective memoryformation continue to court after such a training as vigorously,as naïve males.The courtship index was calculated for each male. The learning index (LI)was computed according to the formula:Analysis of spatial nuclear organizationdelimited to FECs formedby the X chromosome region 11ABBerlin, ts3agn and their hybrids are presented in Table 1. Thecolumns 1–4 show the pattern of FECs between the 11AB regionand other X chromosome regions pertinent to listedstrains. The absence of significant differences of FECs betweencolumns, i.e. 1 vs 2 and 3 vs 4 indicates matroclinicinheritance, 2 vs 3 indicates hybrid-specific frequencies, 2 vs 4and 1 vs 3 pinpoints the patroclinic inheritance. The polytenechromosome bands in hybrids demonstrate differences inFECs, a part of them showing either matroclinic propertiesor properties of the father strain. In certain bands, FECs aresimilar in reciprocal hybrids or depend on the direction of a cross, but significantly differfrom that of parents.Spatial nuclear organization was analyzed using microscopicimages of polytene chromosomes in larvae salivaryglands. The results of comparative analysis of FECs betweenthe 11AB region and the other X chromosome regions in Berlin, ts3agn and their hybrids are presented in Table 1. Thecolumns 1–4 show the pattern of FECs between the 11AB regionand other X chromosome regions pertinent to listedstrains. The absence of significant differences of FECs betweencolumns, i.e. 1 vs 2 and 3 vs 4 indicates matroclinicinheritance, 2 vs 3 indicates hybrid-specific frequencies, 2 vs 4and 1 vs 3 pinpoints the patroclinic inheritance. The polytenechromosome bands in hybrids demonstrate differences inFECs, a part of them showing either matroclinic propertiesor properties of the father strain. In certain bands, FECs aresimilar in reciprocal hybrids or depend on the direction of a cross, but significantly differfrom that of parents.NCBI Genome Data Viewer software helped to reveal thegenes located in the X chromosome bands forming the ectopiccontacts. Based on the assumption that shared location ofgenes determines their functional features ,it was worth elucidating what biological processes are underthe influence of epigenetic factors related to allelic parentof-origin. Therefore, the grouping of genes implied theirinvolvement in control of a certain biologic process .ts3agn×Berlin relative to the reciprocalhybrid are genes for motor proteins  and sensoryreceptors . Genes involved in neurodevelopment,oxidative-reduction process and proliferation do not present inBerlin×ts3agn progeny. Heterosis, when the number of genesinvolved in hybrid-specific contacts is more than 2-fold higherthan in both parents, is manifested for oxidative-reductionprocess (3), signal transduction (4), proliferation (9) and noncodingRNAs (13).Figure 3, a presents provisional definition of the biologicalprocesses controlled by genes within bands forming contactswith 11AB region either in matroclinic or in reciprocal hybridspecificmanner. Among the functional groups of genes havingincreased number in ts3agn×Berlin relative to thereciprocal hybrid), reactive oxygen species metabolic process, and metabolism . Sensory perception andsignal transduction groups do not present in Berlin×ts3agn.The biological processes and genes involved in ectopiccontacts with 11AB in a mode of father strain are shown inFig. 3, b. Among them are the genes responsible for chromatinremodeling ; metabolism ; cell proliferation ); chromatine remodeling . Reparation/recombination group does not present in Berlin×ts3agn.Data on biological processes and the number of genes involvedin contacts with 11AB region exclusively in hybridsor with FECs prevailing those of parents (FEC heterosis), areshown on Fig. 3, c. The ratio of gene numbers for Profiles of the localized fragment frequencies (LFFs)Using our specially designed software Homology SegmentAnalysis, we analyzed correlation between ectopic pairingand distribution of small identical fragments within contactingregions.The region X:11AB (~790 kb) involved in DrosophilaX – X:11AB ectopic pairing was selected as the source ofsmall 30 nt fragments, and the profile of X:11AB localizedfragment frequencies (LFFs) was constructed for the X chromosome. The region X:14B–15B was selected asa control region having almost equal length (~790 kb).ts3agn×Berlin FEC (X:11AB). Probably,20 nt fragments are too short to display DNA homologyspecifically associated with ectopic pairing. The increasein fragments length from 20 to 50 nt leads to decrease inLFF (X:14B–15B) – FEC (X – X:11AB) unspecific correlation and simultaneous increase in LFF – FEC (X:11AB) specificcorrelation. However, there is a lack of LFF (50 nt) – ECFcorrelation for ts3agn. Hence, 30 nt fragment length seems tobe optimal in search for the identical fragments within thecandidate chromosomal regions involved in ectopic pairing.Both LFF and the FEC (X – X:11AB) distributions significantlydiffer from normal. Therefore, we calculated Spearman’srank-order correlation coefficients (rS) for LFF andthe strain-specific FEC (X – X:11AB) (Table 2). A positivecorrelation between LFF and FEC is observed within the region of interest X:11AB. The influence of the fragment lengthon rS value has been tested. For 30 nt fragments, rS is highlysignificant for all Drosophila strains ( p < 0.001). As to LFF(X:14B–15B), there is no significant correlation with FEC(X – X:11AB) ( p ≥ 0.01) pointing to specificity of analysisof DNA homology within the region of ectopic pairing. For 20 nt fragments, there is a false-positive correlation betweenLFF (X:14B–15B) and Berlin FEC and thelocalization frequencies for the several specific X:11AB 30 ntfragments having the highest number of occurrences (NO)in the X chromosome (Table 3). The maximal correlation isevident in the part of 372 bp middle repetitive DNA sequence). The majorityof fragments with significant positive rS values appear to bethe parts of a ~50 bp repeat (marked with #) having significantself-complementarity. This sequence also shows an almostcomplete identity to another part of 372 bp repeat. Such repeatswith slight sequence variations occur in both DNA strands ofthe X chromosome. rS is much lower for (-at-)15 repeat and isnearly absent for (-gt-)15 (-tc-)15 and (-ca-)15 repeats, althoughtheir NO can be higher. Noteworthy, all rS values for 372 bprepeat fragments are lower compared to rS for the whole setof the localized fragments (see Table 2), hence all of themseemingly impact ectopic pairing.To find out what DNA sequences impact the ectopic pairingthe most, we calculated rS between miRNAs expression profileBerlin, ts3agn and their reciprocalhybrids demonstrates significant differences betweenmales of parent strains and hybrids in content of 44 miRNAs.For the reciprocal hybrids, the heat maps of miRNAs expressionare presented in Fig. 5. Among 44 miRNAs 10 miRNAsbelong to the same cluster of testis-specific miRNAs . However, only miR-980 and miR-974are involved in memory processes. Expression of miR-980suppresses Drosophila memory ,while lowered expression of miR-974 impairs memory formationAnalysis of miRNAs expression in The heat map represents the RPM-normalized and log2-transformed counts of miRNAs reads with z-scale normalizationof the rows. Thirty percent of low-expressed miRNAswere removed from further analysis. Only the miRNAs withaltered content in a hybrid compared to at least one parentare shown. analysis of parent strainsand their reciprocal hybridsLocomotor behavior. Figure 6 presents the parameters oflocomotion. As to activity indices, they are significantly differentin ts3agn and Berlin, both hybrids differ from ts3agn andBerlin×ts3agn differs from Berlin. Running speed and runfrequency in ts3agn and Berlin are similar. Both hybrids differfrom ts3agn and Berlin×ts3agn from Berlin. However, thehybrid running speed exceeds that of parents, demonstratingheterosis. Comparatively to parents, run frequency in hybridsis intermediate. At the same time, any alterations in locomotorparameters in hybrids are similar and unidirectional.Learning acquisition and memory formation.In all strains,CIs of males decrease after training with fertilized femalescompared to naïve flies . Learning/memory scoresin reciprocal hybrids show that memory formation (3 hoursafter training), but not learning acquisition (0 hours aftertraining), demonstrates patroclinic inheritance .Berlin strain in crossests3agn×Berlin, learning and 3-hour memory do not differfrom LIs of Berlin, but do differ from LIs of ts3agn. Patroclinicinheritance is evident in the reciprocal hybrid Berlin×ts3agn.In this case, 3-hour LIs in Berlin×ts3agn and ts3agn are similar.The patroclinic inheritance cannot be associated with theY chromosome, as it is likely to be similar in Berlin, ts3agnand Canton S . Also, it cannot be attributed to the X chromosomes,since they are different in Berlin and ts3agn×Berlin.Seemingly, this may be caused by some paternal epigeneticfactors, such as miRNAs in cytoplasm of male sperm.When male parent originates from Discussionts3agn gene and epigenetic factors (POEs) in spatial nucleararchitecture, learning/memory formation and spontaneouslocomotor activityStudies of genome-wide associations between DNA polymorphismsand phenotypic traits have revealed genetic variantspredisposing to different mental diseases. Findings pinpointingthe role of POEs in genetic risk for neuropathology opennew possibilities for therapy and preventive medicine . In this study, we estimated FECs in reciprocalhybrids considering an impact both of genetic variants ofdlimk1 gene.Our assumption was that FEСs partly reflect the restrictedhomology of short DNA sequences in different, seemingly“non-homologous” regions.To exploit the advantages given by the model, we delimitedthe analysis of spatial nuclear organization to FECs formedby the X chromosome region 11AB harboring As shown in this study for the short identic fragments withinthe contacting regions, FECs correlate with LFFs. Althoughthe correlation is rather moderate (~0.35), it is highly specificfor 30 nt fragments. Many factors affect ectopic pairing,such as DNA homology, the distance between the interactingregions and epigenetic factors causing the interstrainFECs differences . Our computationalalgorithm concerns only fragments aligned with the X chromosomewithout gaps. This reveals the partial homology ofinteracting bands. Thus, the interacting chromosomal areasmay be significantly larger than 30 or 50 nts. Although differentmechanisms are involved in ectopic pairing, includingPOEs, our data indicate the significant role of DNA sequenceitself. However, as FEC–LFF correlation is mainly observedfor specific DNA fragments, their pairing mediated by someproteins or non-coding RNAs cannot be ruled out.Most of the found 30–50 nt fragments are similar to theD. melanogaster dispersed 372 bp A/T-rich noncoding repeat. This moderately repeated sequence islocated in the euchromatin of the X chromosome between theregions 4 and 14A in ~300–400 copies per haploid genome.The 372 bp repeat is a part of 1.688 g/cm3 class of satelliteDNA (1.688X repeats) . siRNAfrom the 1.688X repeats is involved in dosage compensationin recognition of the X and autosomal chromatin, therebydelimiting activities of male-specific lethal (MSL) complexto sex chromosomes through up-regulation of the X chromosome.ts3agn, FECs in thesebands significantly decrease. In this case maternal control ofspatial localization and therefore, of gene expression is geneticallydetermined. These are genes controlling membranereceptor regulation (PPYR1) and signal transduction (gce),chromatin remodeling , axon guidance andchemosensory jumping behavior (acj6 ).The data obtained consider the common mechanisms ofectopic contacts formation and dosage compensations. Seemingly,POEs might influence the spatial chromatin organization,thereby affecting behavioral performances.This indicates that each Drosophila strain possesses itsown pattern of ectopic contacts with the region 11AB. Thepolytene chromosome bands are heterogeneous in their modesof regulation of ectopic pairing. Apart of them is regulated bygenes of either maternal, or paternal origin. A separate classis comprised of regions manifesting only hybrid properties.Similar РОЕs were observed for the pattern of methylation andnucleosome distribution within the imprinted loci in humansand plants .In both reciprocal crosses, bands 7A, 9A and 13B displaythe maternal properties. When mother is This indicates that each Drosophila strain possesses itsown pattern of ectopic contacts with the region 11AB. Thepolytene chromosome bands are heterogeneous in their modesof regulation of ectopic pairing. Apart of them is regulated bygenes of either maternal, or paternal origin. A separate classis comprised of regions manifesting only hybrid properties.Similar РОЕs were observed for the pattern of methylation andnucleosome distribution within the imprinted loci in humansand plants .ts3agn, FECs in thesebands significantly decrease. In this case maternal control ofspatial localization and therefore, of gene expression is geneticallydetermined. These are genes controlling membranereceptor regulation (PPYR1) and signal transduction (gce),chromatin remodeling , axon guidance andchemosensory jumping behavior (acj6 ).In both reciprocal crosses, bands 7A, 9A and 13B displaythe maternal properties. When mother is ts3agn×Berlin, the number of genes with knownfunctions contacting with 11AB with maternal-specific frequencyis 2-fold higher than in reciprocal cross. Possibly, thisis due to the ts3agn-specific miRNAs pattern of expression.The role of miRNAs in maternal inheritance and expressionin embryogenesis is sparsely studied. As we have shownearlier, the expression level of miR-9, miR-34 and miR-124differs in ts3agn from that in Berlin, Canton S and Oregon-R. miR-9 and miR-124 are alsoexpressed in early development (0–12 hrs) , miR-34 is detected in embryos till zygotic reduction. As known, the switch from maternal tozygotic development program occurs between the second andthe third hours of embryonic stage, hence miRNA found inearly development have maternal origin . ThesemiRNAs targets are Swi/Snf-like complex, neural-progenitorspecificnpBAF, repressor-element-1-silencing transcriptionfactor (REST belonging to 1 class of histone deacetylases(HDAC1/2) and silent information regulator 1 (SIRT1) –3 class of NAD+-dependent histone deacetylases involved inheterochromatin formation, Bourassa, Ratan, 2014). They areinvolved in neurogenesis, dendrite morphogenesis and axonguidance which depend on global chromatin remodeling.In the cross ts3agn×Berlinectopic contacts between 11AB and regions containing genesinvolved in chromosome remodeling are formed with frequencycharacteristic for the maternal genome. The products ofthese genes are: Tip60 – histone acetyltransferase, HDAC6 –histone deacetylase; mxc – regulator of histone synthesis ofPolycomb group; Top1 – DNA topoisomerase. However, onlytwo regions containing genes HDAC6 and Top1 are presentin cross Berlin×ts3agn.Therefore, it is not surprising that in cross ts3agn-like matroclinicmode of inheritance is pertinent to genes responsible for actinand microtubules-binding proteins with motor function andneurodevelopment. Noteworthy, the state of actin remodelingdetermining neurologic manifestations is a diagnostic featureof ts3agn .Noteworthy, new knowledge about topologically associatingdomains (TADs) indicates that polytene, diploid, andembryonic TADs condensation along the chromosome axisis just the same everywhere . Moreover,comparison of TADs with 3D chromatin organization revealedby the Hi-C method confirms that the interphase nucleusspatial organization into TADs is directly represented by bandingpattern of polytene chromosomes .Therefore, this allows to bridge the ratio of genes forming ectopic contacts in a mode of either maternal, or paternal strainin reciprocal crosses and their physiologic manifestations.The later might result from alterations in the 11AB regionarchitecture. As shown in Fig. 3, a, the The regions with FECs similar in reciprocal hybrids, butdiffering from parents, i. e. manifesting hybrid properties,contain a large set of genes responsible for motor functions.ts3agn:meiosis, reparation, recombination; transcription factors; metabolism;proliferation; actin-binding proteins, microtubuleassociatedproteins.Figure 3, c shows genes and biological processes for chromosomalregions forming ectopic contacts with X:11AB onlyin hybrids or mainly in hybrids compared to parents. Theseprocesses are pertinent to main manifestations of ts3agn the chromosomalbands 8D, 12E, and 19D demonstrate FECs significantly exceedingthese of parents. These bands contain genes involvedin taste and odor perception and neurodevelopment, in particularof the mushroom bodies of the brain. The other examplesof father strain manifestations might result from activities oftrans-acting factors, such as miRNAs .Interestingly, in the cross Berlin×ts3agn and in progenyof Berlin×ts3agn (impaired 3-hour memory) and is similar towild type in the ts3agn×Berlin cross . Likely,miR-974 might act as trans-acting factor presumed to regulategenes in patroclinic mode. The prevailing role of the paternalgenome in memory formation is evident in Canton S and ts3agnreciprocal hybrids .miR-974 is involved in memory processes: its loweredexpression impairs memory formation .Decrease in its content in olfactory neurons and the mushroombody V2 neurons promotes 3-hour memory. Noteworthy, thecontent of miR-974 is decreased both in agnostic locusmight belong to the class of quantitative trait loci (QTL) .Taken together, our data indicate that the agnostic LIMK1 gene is a good candidate for linkingthe neuronal activity  and genetic apparatus . Additionally, quite recent andunexpected findings  reveal a new targetof intellectual disabilities: learning acquisition and memory erasure (forgetting) are governed by different signal cascades,correspondently cAMP-dependent and actin remodeling cascadesmall GTPase Rac1 – LIMK1 (the key enzyme of actinremodeling LIM-kinase 1) and its phosphorylation substrateсofilin. The absence of Rac1-dependent forgetting causesthe autistic spectrum disorders. Expression changes (activeor non-active state) of LIMK1 and cofilin lead to differentneurological disorders. Therefore, in the tradition of Russiangenetic school , the agnostic genemight be a functional link between genetic and cytogeneticprocesses within the nervous system and serve as a model forelucidating both the maternal and paternal modes of transgenerationalinheritance.One of the requirements of predictive and personalizedmedicine is consideration of POEs for prognosis of clinicalphenotype of many multifactorial neuropsychiatric disorders.These different and individual manifestations of cognitiveabilities and motor functions in patients with the same disease,i. e. behavioral plasticity, results from genome plasticity provokedby 3D chromatin architecture of the nerve cells nuclei.The evolutionary gene conservation approves the usage ofsimple low cost, fast and efficient models as Drosophila toprobe the causes, consequences and mechanisms of pathologyleading to human disease . 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Antenatal care is a unique opportunity to assess SARS-CoV-2 seroprevalence and antibody response in pregnant people, including those with previously unknown infection. Pregnant people were screened for SARS-CoV-2 IgG during antenatal care or delivery in Seattle, Washington with Abbott Architect chemiluminescent immunoassay which provides quantitative index (positive ≥1.4). Participants with IgG+ results or identified with RT-PCR+ results via medical records were invited to enroll in a longitudinal evaluation of antibody responses. We report preliminary results of an ongoing seroprevalence and longitudinal study with planned 18-month follow-up.≤280 days after first RT-PCR+ result for those with and ≥104 days after first IgG detection for those without prior RT-PCR+ results), while 5/14 (26%) had a negative Abbott IgG test at a median of 81 days (IQR 75–112) since initial testing.Between September 9, 2020–May 7, 2021, we screened 1304 pregnant people; 62 (4.8%) tested SARS-CoV-2 IgG+, including 28 (45%) with known prior SARS-CoV-2 infection. Among participants testing IgG+, median age was 32 years (interquartile range [IQR] 26–35) and median gestational age was 21 weeks (IQR 12–38) at screening; median IgG index was 3.2 , including 3.9 (IQR 2.3–5.8) among those with vs. 2.7 (IQR 1.9–4.2) among those without prior RT-PCR+ results (p=0.05 by Wilcoxon rank-sum). Of 30 longitudinal study participants enrolled, 24 tested IgG+ at baseline (75% with prior RT-PCR+ result) and 6 tested IgG- on enrollment but were identified as previously RT-PCR+ via medical records; 24/30 (80%) reported previous symptoms. Of 24 participants testing IgG+ at baseline, 14 (58%) had first follow-up IgG results at median of 66 days (IQR 42–104) since initial testing, with median IgG index of 2.0 (IQR 1.0–3.8). 9/14 (64%) participants with repeat IgG testing remained IgG+ at first follow-up  Alisa Kachikis, MD, MS, GlaxoSmithKline (Consultant)Pfizer (Consultant) Alexander L. Greninger, MD, PhD, Abbott (Grant/Research Support)Gilead (Grant/Research Support)Merck (Grant/Research Support) Janet A. Englund, MD, AstraZeneca GlaxoSmithKline (Research Grant or Support)Meissa Vaccines (Consultant)Pfizer (Research Grant or Support)Sanofi Pasteur (Consultant)Teva Pharmaceuticals (Consultant) Alison Drake, PhD, MPH, Merck (Grant/Research Support)


The cirrhosis-associated abnormalities of ACE, IL-6, VWF antigen, and antiplasmin parallel those observed in severe COVID-19.(1) Background: Cirrhotic patients have an increased risk for severe COVID-19. We investigated the renin-angiotensin-aldosterone system (RAS), parameters of endothelial dysfunction, inflammation, and coagulation/fibrinolysis in cirrhotic patients and in COVID-19 patients. (2) Methods: 127 prospectively characterized cirrhotic patients (CIRR), along with nine patients with mild COVID-19 (mild-COVID), 11 patients with COVID-19 acute respiratory distress syndrome , and 10 healthy subjects (HS) were included in the study. Portal hypertension (PH) in cirrhotic patients was characterized by hepatic venous pressure gradient (HVPG). (3) Results: With increased liver disease severity (Child−Pugh stage A vs. B vs. C) and compared to HS, CIRR patients exhibited higher RAS activity (angiotensin-converting enzyme (ACE), renin, aldosterone), endothelial dysfunction (von Willebrand-factor (VWF) antigen), inflammation (C-reactive protein (CRP), interleukin-6 (IL-6)), and a disturbed coagulation/fibrinolysis profile . Increased RAS activity (renin), endothelial dysfunction (vWF), coagulation parameters  and inflammation  were significantly altered in COVID patients and followed similar trends from mild-COVID to ARDS-COVID. In CIRR patients, ACE activity was linked to IL-6 (ρ = 0.26;  Severe acute respiratory distress syndrome-coronavirus-2 (SARS-CoV-2) causes substantial morbidity and mortality worldwide . CoronavPatients with ARDS due to COVID-19 exhibit severe endothelial damage, pulmonary microangiopathy, and thrombosis ,5, indicSimilarly, coagulation imbalance, inflammation, and RAS activation also represent well-known hallmarks of advanced chronic liver disease (ACLD) ,12,13. CPatients with ACLD are at particularly high risk for severe courses of COVID-19 ,17; howeACLD patients undergoing hepatic venous pressure gradient (HVPG) measurement at the Vienna General Hospital between February 2019 and December 2020 with portal hypertension (CIRR) were included in this study. Hemodynamic parameters including mean arterial pressure (MAP) were assessed at the time of HVPG measurement. Blood samples were withdrawn after the patients had rested for at least 30 min in supine position.Patients with intake of non-selective beta-blockers, antithrombotic or antiplatelet therapy, with active malignancy, portal vein thrombosis, porto-sinusoidal vascular disease or cardiac cirrhosis, as well as patients after liver transplantation were excluded from the study. If multiple HVPG measurements were performed in the same patient, only the baseline measurement  was used for this study. The ACLD patients were stratified by Child−Turcotte−Pugh (CTP) stage A, B, and C and by HVPG . Etiology of ACLD, comorbidities , age, and intake of concomitant medication were recorded.Moreover, inpatients at the General Hospital of Vienna with COVID-19 (mild-COVID), inpatients with ARDS due to COVID-19 (ARDS-COVID), as well as outpatients without ACLD or COVID-19  willing to participate in the study were included.For HVPG measurement, the right jugular vein was accessed via Seldinger technique using a catheter introducer set . A balloon catheter  was subsequently used for liver vein cannulation . HVPG waA colorimetric assay  was used to determine ACE activity. Plasma concentrations of renin  and aldosterone  were measured by chemiluminescence-immunoassay. VWF antigen was measured by latex agglutination assay . Prothrombin fragment F1,2 and plasminogen activator inhibitor (PAI) were assessed by ELISA , plasminogen activity, and α-2 antiplasmin (antiplasmin) activity by chromogenic assay . Routine laboratory parameters including D-dimer, C-reactive protein (CRP), and interleukin-6 (IL-6) were assessed by standard laboratory methods.Categorical variables were presented as number (n) of patients and % of these patients with the characteristic of interest, while continuous data was reported as median and interquartile range (IQR). D’Agostino and Pearson and Shapiro–Wilk normality tests were used to test for normal distribution. Mann-Whitney U test was implemented for comparing non-normally distributed continuous variables between two groups, and Kruskal-Wallis test for comparison of non-normally distributed continuous variables in three or more groups. Dunn’s multiple comparisons test was used as post-hoc test. Group comparisons of categorical variables were computed using Pearson’s Chi-squared or Fisher’s exact test.p < 0.100) were included in the multivariate model. Multicollinearity was investigated via variance inflation factor (VIF). GraphPad Prism 8  and IBM SPSS 22.0 statistic software  were used for statistical analysis. A two-sided p-value of <0.05 was considered as being statistically significant.Factors associated with ACE plasma levels were assessed using linear regression models including either VWF antigen, prothrombin fragment F1,2, or antiplasmin activity. Parameters that showed a trend  (NCT03267615).The study was approved by the ethics committee (EC) of the Medical University of Vienna (study number: 1461/2020 and 1262/2017). It was performed according to the current version of the Helsinki Declaration. All patients were prospectively included in the study and gave their informed consent before the blood withdrawal. The cirrhotic cohort of this study is part of the Vienna Cirrhosis Study registry, registered under p < 0.001).In total, 127 CIRR patients with male predomination  were included in the study. Median age was 56.6 (IQR 15.5) years. Alcoholic (49.6%), viral (15.0%), and cholestatic liver disease (8.7%) were the main etiologies . There wThe mild-COVID cohort consisted of nine patients with COVID-19, who were admitted to the Vienna General Hospital due to the viral infection , while the ARDS-COVID cohort included 11 patients  and the HS cohort included 10 subjects  .p < 0.001) and VWF antigen (A: 223.4 (IQR 114.0)% vs. B: 307.0 (IQR 133.0)% vs. C: 396.0 (91.0)%; p < 0.001) significantly increased with decreasing liver function, while there was a trend for PAI levels (A: 0.89 (1.60) IU/mL vs. B: 0.92 (2.06) IU/mL vs. C: 2.68 (1.73) IU/mL; p = 0.149) (p < 0.001), plasminogen activity (A: 75.0 (21.0)% vs. B: 59.0 (12.0)% vs. C: 42.0 (9.0)%; p < 0.001), and in tendency prothrombin fragment F1,2 (A: 287.0 (292.0) pmol/L vs. B: 292.5 (241.0) pmol/L vs. C: 176.0 (194.0) pmol/L; p = 0.079) decreased with increasing CTP stage (D-dimer (A: 0.48 (IQR 0.41) µg/mL vs. B: 1.40 (IQR 2.90) µg/mL vs. C: 3.19 (3.08) µg/mL; = 0.149) . AntiplaTP stage . SimilarTP stage .p < 0.001) (p < 0.001). Similarly, more pronounced RAS activation was observed in patients with more advanced hepatic dysfunction: ACE (A: 41.3 (35.9) U/L vs. B: 54.4 (45.6) U/L vs. C: 71.3 (66.9) U/L; p = 0.006), as well as plasma renin (A: 12.4 (26.7) µIU/mL vs. B: 55.1 (115.9) µIU/mL vs. C: 594.1 (949.8) µIU/mL; p < 0.001) and aldosterone concentrations (A: 84.5 (442.0) pg/mL vs. B: 240.0 (442.0) vs. C: 397.0 (238.0) pg/mL; p < 0.001).With increasing CTP stage, there was increased inflammation, as indicated by rising CRP (A: 0.18 (0.29) mg/dL vs. B: 0.47 (0.67) mg/dL vs. C: 0.47 (1.23) mg/dL; < 0.001)  and IL-6p = 0.009), vWF antigen (mild-COVID: 247.5 (73.0) vs. ARDS-COVID: 420.0 (99.0)%; p = 0.007), D-dimer (mild-COVID: 0.54 (0.58) µg/mL vs. ARDS-COVID: 2.94 (4.75) µg/mL; p < 0.001), prothrombin fragment F1,2 (mild-COVID: 151.5 (252.0) pmol/L vs. ARDS-COVID: 429.0 (2687.0) pmol/L; p = 0.006), CRP (mild-COVID: 0.50 (1.42) mg/dL vs. ARDS-COVID: 15.10 (14.69) mg/dL; p < 0.001), and IL-6 (mild-COVID: 7.66 (14.74) pg/mL vs. ARDS-COVID: 65.70 (357.90) pg/mL; p < 0.001), indicating an increased RAS activation, endothelial dysfunction, coagulation/fibrinolysis activation, and inflammation  µIU/mL vs. ARDS-COVID: 62.7 (107.1) µIU/mL; ammation .Compared to healthy control subjects, cirrhotic patients and COVID-19 patients exhibited markedly increased VWF antigen, while the highest median VWF antigen level occurred in ARDS-COVID patients . Both ciMoreover, compared to healthy subjects, cirrhotic and COVID-19 patients had higher levels of CRP and IL-6.On the other hand, cirrhotic patients exhibited significantly lower antiplasmin activity and plasminogen activity than COVID-19 patients, while ACE activity and plasma aldosterone concentration were higher.p < 0.001), D-dimer , prothrombin fragment F1,2 , antiplasmin activity , IL-6 , and HVPG   .p = 0.001), coagulation , and of fibrinolysis , but not with parameters of inflammation   .In this study, we thoroughly characterized the state of components of the RAS, coagulation, and inflammation in cirrhotic patients of different disease severity and compared the findings to patients with mild COVID-19 and with COVID-ARDS. Importantly, we identified profound abnormalities of the coagulation system and upregulated systemic inflammation linked to cirrhosis-associated RAS activation that followed similar trends in COVID-19.By stratifying our ACLD cohort by hepatic dysfunction , as well as for portal pressure (by the diagnostic gold-standard HVPG), we observed marked endothelial dysfunction  and dysbalanced coagulation state  with increasing ACLD severity. At the same time, inflammatory markers , along with RAS components, were elevated with more severe ACLD, indicating a state of coagulation/fibrinolysis activation linked to RAS activity—that reportedly is upregulated in cirrhosis ,22.COVID-19 patients exhibited very similar levels of endothelial dysfunction, coagulation/fibrinolysis, and inflammation parameters, compared to cirrhotic patients, as well as RAS activation, indicated by increased plasma renin concentration. Interestingly, these parameters were also the most important factors differing between mild-COVID and ARDS-COVID. This suggests that cirrhotic patients are in a disadvantageous position when contracting SARS-CoV-2 infection. Importantly, the pronounced RAS activation causing associated coagulation imbalance and a proinflammatory state in Child−Pugh B/C patients may explain why cirrhotic patients have been reported to be particularly susceptible to severe courses of COVID-19 ,17.The RAS is intricately involved in cirrhosis development and progression . ImportaIn COVID-19, especially the non-classical RAS is subject of scientific interest, as SARS-CoV-2 uses the ACE2 receptor for cell entry  and it hCOVID-19 is strongly linked to thromboembolic complications, including microvascular thrombosis, but also macrovascular events such as stroke or pulmonary embolism ,30. EndoSimilarly, D-dimer and prothrombin fragment F1,2 were also increased both in cirrhosis and COVID-19, signifying an activation of coagulation and fibrinolysis, with the highest values in ARDS-COVID patients. Interestingly, prothrombin fragment F1,2 levels were highest in Child-A patients, indicating coagulation dysregulation already in an early stage of cirrhosis. Elevated D-dimer was identified as a marker of poor prognosis in patients with COVID-19 . The higFurthermore, high plasmin levels are observed in liver cirrhosis, likely due to decreased antiplasmin and elevated tissue-type plasminogen activator activity . ImportaFinally, both liver cirrhosis and COVID-19 were associated with increased systemic inflammation. As an infectious disease, COVID-19 triggers a complex immune response, leading to a pronounced (and sometimes excessive) inflammatory reaction . Again, Elevation of inflammatory parameters in cirrhosis may be due to gut-derived bacterial translocation ; howeverOur study has limitations: The cohorts were not age- and sex-matched. While cirrhotic patients were mainly male, the COVID-19 cohort was mainly female. Moreover, the healthy control group was significantly younger than the other groups, which may have impacted laboratory tests such as VWF, which increases with age . HoweverIn conclusion, we comprehensively investigated the role of the RAS, endothelial dysfunction, coagulation, and inflammation both in patients with cirrhosis and with COVID-19. Importantly, we found striking similarities between cirrhotic patients and COVID-19 patients, as both groups suffer from endothelial dysfunction, coagulation/fibrinolysis activation, and a systemic pro-inflammatory state. The “baseline-upregulation” of the RAS in the setting of advanced cirrhosis may facilitate SARS-CoV-2 cell entry due to decreased antiplasmin activity and consecutively elevated plasmin levels. The molecular mechanisms driven by cirrhosis-associated RAS activation—including the induction of an imbalanced coagulation profile, endothelial dysfunction, and systemic inflammation—may mechanistically explain a susceptibility to severe COVID-19 in cirrhotic patients.


Correction to: BMC Health Serv Res 21, 733 (2021)https://doi.org/10.1186/s12913-021-06757-x7,8 to D. Laplanche7.Following publication of the original article , the affThe author affiliation list has been updated above and the original article  has been


Cohort studies suggest higher rates of discontinuations (DCs) and adverse events (AEs) with integrase inhibitors (INSTIs) than is reported in clinical trials. Here, we assess DC of different INSTIs in combination with one of two tenofovir prodrugs in the first year following initiation defined as “early DC” in a real-world cohort of treatment-naïve patients.This analysis evaluated treatment-naïve patients at a single center initiating raltegravir (RAL), elvitegravir/cobicistat (EVG/c), dolutegravir (DTG) or bictegravir (BIC) in combination with emtricitabine/tenofovir alafenamide (F/TAF) or emtricitabine/tenofovir disoproxil fumarate (F/TDF) between 10/2007-1/2020. Eligible patients had a minimum follow-up of 1 year. The primary endpoint was incidence of early INSTI DC. Secondary endpoints included AEs and risk factors for early INSTI DC and treatment-related AEs.331 patients were included. Median age was 32 years, 89% were male, 43% were non-White, 8% started RAL-based therapy, 46% started EVG/c-based therapy, 22% started DTG-based therapy and 24% started BIC/F/TAF. 36 discontinued INSTI-based therapy early yielding an incidence rate of 0.17 DCs per person-years (PPY) among RAL patients, 0.14 DCs PPY among EVG/c patients, 0.22 DCs PPY among DTG patients, and 0 DCs PPY among BIC patients, p=0.006. Treatment-related AEs occurred in 27% of RAL patients, 42% of EVG/c patients, 50% of DTG patients, and 43% of BIC patients p=0.607; and were responsible for early DC rates of 0.022 in 3 EVG/c patients and 0.075 in 5 DTG patients. No treatment-related early DCs occurred among RAL or BIC patients. No evaluated factor was significantly associated with early INSTI DC, however DTG use was significantly associated with treatment-related AEs .Table 1. Risk factors for early integrase inhibitor discontinuation and treatment-related adverse eventsIn this cohort, early DCs occurred in 11% initiating INSTI-based therapy, however of these only 2% were treatment-related. These data support use of INSTI-based regimens as preferred options for treatment-naïve patients living with HIV due to their favorable safety and tolerability profiles.Charlotte-Paige M. Rolle, MD MPH, Gilead Sciences Janssen Infectious Disease ViiV Healthcare  Kiran Patel, PharmD, Gilead Sciences (Employee) Federico Hinestrosa, MD, AbbVie (Speaker’s Bureau)Gilead Sciences Theratechonologies (Advisor or Review Panel member)ViiV Healthcare  Edwin DeJesus, MD, Gilead Sciences


Klebsiella pneumoniae is an increasingly important hospital pathogen. Classical K. pneumoniae (cKp) and hypervirulent K. pneumoniae (hvKp) are two distinct evolutionary genetic lines. The recently ongoing evolution of K. pneumoniae resulted in the generation of hybrid hvKP-MDR strains. K. pneumoniae distinct isolates (n = 70) belonged to 20 sequence types with the prevalence of ST395 (27.1%), ST23 (18.6%), ST147 (15.7%), and ST86 (7.1%), and 17 capsular types with the predominance of K2 (31.4%), K57 (18.6%), K64 (10.0%), K1 (5.7%) were isolated from patients of the Moscow neurosurgery ICU in 2014–2019. The rate of multi-drug resistant (MDR) and carbapenem-resistant phenotypes were 84.3% and 45.7%, respectively. Whole-genome sequencing of five selected strains belonging to cKp (ST395K47 and ST147K64), hvKp (ST86K2), and hvKp-MDR (ST23K1 and ST23K57) revealed blaSHV, blaTEM, blaCTX, blaOXA-48, and blaNDM beta-lactamase genes; acr, oqx, kpn, kde, and kex efflux genes; and K. pneumoniae virulence genes. Selective pressure of 100 mg/L ampicillin or 10 mg/L ceftriaxone induced changes of expression levels for named genes in the strains belonging to cKp, hvKp, and hybrid hvKp-MDR. Obtained results seem to be important for epidemiologists and clinicians for enhancing knowledge about hospital pathogens. Klebsiella pneumoniae is an increasingly important hospital pathogen causing a wide range of infections including urinary tract infections, pneumonia, bacteremia, and liver abscesses. In severe clinical cases, it can also lead to multiple organ failure, or even death [K. pneumoniae (cKp) and hypervirulent K. pneumoniae (hvKp), were described and are both global pathogens [K. pneumoniae strains belong to particular clones  producing beta-lactamases in combination with other functional classes of resistance determinants [K. pneumoniae were attributed to sequence types ST23, ST86, ST65, etc., and capsular types K1, K2, K57, K20, etc. [en death . Two difathogens . Most muK. pneumoniae resulting in the generation of hybrid hvKp-MDR strains. Mechanisms for the emergence of such strains can be a result of acquiring hypervirulent plasmids by cKp [Recent studies have shown the ongoing evolution of s by cKp , acquiris by cKp , and acqs by cKp . HoweverK. pneumoniae strains collected in Moscow neurosurgery ICU in 2014–2019, to identify resistance and virulence genes in their cells, and to estimate relative expression levels of such genes in selected strains belonging to epidemiology significant genetic lines cKp (ST395K47 and ST147K64), hvKp (ST86K2), and hybrid hvKp-MDR (ST23K1 and ST23K57).This study aimed to determine the genetic lines of K. pneumoniae caused about 28% among the agents of nosocomial infections in neurosurgery ICU during the period from January 2014 to May 2019. The incidence rates of K. pneumoniae infections were 8.0 per 100 patient infections of the central nervous system, 4.3/100 of bloodstream infections, 26.3/100 of respiratory infections, and 25.3/100 of urinary tract infections [K. pneumoniae clinical isolates were collected from 283 patients in this period, including those isolated from the respiratory system (n = 271), urine (n = 166), the nervous system (n = 41), blood (n = 36), surgical wounds (n = 27), and other (n = 4).n = 34), urine (n = 19), the nervous system (n = 8), blood (n = 6), and surgical wounds (n = 3). As a result, 20 sequence types were identified, the majority of them were ST395, ST23, ST147, and ST86, and a total of 17 capsular types were identified. Predominant K-types were K2, K57, K64, and K1 , ampicillin-sulbactam (90.0%), cefuroxime (86.8%), cefoxitin (68.8%), ceftriaxone (77.8%), ceftazidime (72.8%), cefoperazone-sulbactam (82.7%), cefepime (69.2%), ertapenem (48.1%), tetracycline (80.7%), ciprofloxacin (81.2%), chloramphenicol (77.0%), gentamicin (67.1%), tobramycin (83.0%), trimethoprim-sulfamethoxazole (69.2%), and nitrofurantoin (82.2%). Fewer resistant isolates were found for imipenem (33.8%), tigecycline (37.2%), and amikacin (31.8%) (It was shown that major  (31.8%) .K. pneumoniae isolates carried beta-lactamase genes blaSHV (100.0%), blaCTX-M (74.2%), blaTEM (51.4%), blaOXA-48 (40.0%), blaNDM (11.4%), class 1 (37.6%) and 2 (2.1%) integrons, and porin protein gene ompK36 (91.5%). Both blaOXA-48 and blaNDM carbapenemase genes were detected in four (5.7%) isolates. Beta-lactamase genes blaKPC, blaVIM, and blaIMP were not detected (The frequency of the multi-drug resistant (MDR) phenotype was 84.3%, with the carbapenem-resistant phenotype consitituting 45.7%. detected .K. pneumoniae isolates: rmpA (34.2%), aer (21.4%), kfu (17.1%), uge (85.7%), wabG (100.0%), fimH (97.1%), allS (94.3%), and allR (5.7%). Twelve virulence gene combinations were identified. The most prevalent combination was uge+wabG+fimH+allS (51.4% isolates), followed by rmpA+aer+uge+wabG+fimH+allS (11.4%), uge+wabG+kfu+fimH+allS (7.1%), rmpA+wabG+fimH+allS (5.7%), and rmpA+aer+ +uge+wabG+kfu+fimH+allR (5.7%). The rarest combinations were wabG+fimH+allS (4.3%), rmpA+uge+wabG+fimH+allS (4.3%), uge+wabG+allS (2.8%), rmpA+aer+wabG+fimH+allS (2.8%), rmpA+wabG+kfu+fimH+allS (1.4%), rmpA+aer+uge+wabG+kfu+fimH+allS (1.4%), and rmpA+wabG+kfu+fimH+allS (1.4%) (Eight virulence genes were detected in 70 S (1.4%) .n = 9) and urine (n = 2), belonging to genetic lines ST23K1 (n = 4), ST86K2 (n = 3), ST23K57 (n = 2), ST65K2 (n = 1), and ST218K57 (n = 1). Isolates of ST23K1 carried rmpA+aer+uge+wabG+kfu+fimH+allR; isolates of ST86K2 and ST218K57 harbored rmpA+aer+uge+wabG+fimH+allS; isolates of ST23K57 carried two combinations, rmpA+uge+wabG+fimH+allS and rmpA+wabG+kfu+fimH+allS; and isolates of ST65K2 carried rmpA+aer+uge+wabG+kfu+fimH+allS virulence gene combinations. All 11 hypermucoviscous isolates carried rmpA, wabG, and fimH genes. Major isolates of hypermucoviscousity-positive isolates were MDR (n = 6), resistant to 3–7 antimicrobial functional groups . Among them, two isolates were CR. The remaining isolates were resistant to ampicillin only. As a result, there was no revealed correlation between the hypermucoviscousity phenotype and genetic line or the antimicrobial phenotype or virulence gene profiles (Phenotype of hypermucoviscousity was detected for 11 of 70 isolates (15.7%) collected from the respiratory system , hvKp (ST86K2), and hvKp-MDR (ST23K1 and ST23K57), were analyzed by whole-genome sequence and quantitative analyses of antimicrobial resistance and virulence gene expression. One of these strains (B2523/18) was resistant to AMP and carried only one beta-lactamase gene of the blaSHV type. In contrast, the other four strains that belonged to the MDR category were resistant to 10–18 antimicrobials. The latter strains carried beta-lactamase genes of blaSHV, blaTEM, blaCTX-M, blaOXA-48, and blaNDM types and the resistance determinants for other antimicrobial groups in the genomes: aminoglycosides, fosfomycin, streptogramins, phenicols, quinolones, sulfonamide, tetracyclines, sulfonamides, macrolides, and rifamycins, as well as genes coding 4–5 efflux pumps and 1–4 genes of heavy metal resistance. The following genetic determinants of K. pneumoniae virulence were identified in the genomes: peg-344 (n = 3), rmpA (n = 3), rmpA2 (n = 1), iroB (n = 2), iroN (n = 2), iroD (n = 2), uge (n = 5), wabG (n = 5), kfu (n =1 ), fimH (n = 5), allR (n = 1), irp (n = 4), iuc (n = 3), entB (n = 3), iut (n = 4), mrk (n = 5), ybt (n = 4), fyu (n =4 ), treC (n = 5), celB (n = 5), and ureA (n = 5)  .K. pneumoniae strains during growth without selective pressure of antimicrobials were different: the chromosomal beta-lactamase genes blaSHV were transcribed significantly lower compared with those of the reference gene rpoD. In contrast, other beta-lactamase genes , as well as porin gene ompK36 were transcribed at higher levels compared with the reference gene, with the exception of blaTEM in the hvKp-MDR strain of ST23K1 which exhibited lower expression. The efflux pump genes and the virulence genes were expressed mostly at the same or lower levels compared with the rpoD gene, with the exception of two virulence genes: treC in the strains of ST395K47, ST147K64, ST23K1, and ST23K57; and celB in the strain of ST86K2  gene, and downregulation of the blaTEM (5.5-fold), acrA (2.4-fold), acrB (2.8-fold), oqxB (4.8-fold), kpnE (3.5-fold), kpnF (2.0-fold), and kdeA (3.0-fold) genes. Another MDR strain of ST147K64 was characterized by the upregulation of the blaTEM (2.2-fold), kpnE (3.2-fold), kdeA (2.7-fold), fimH (2.3-fold), and ureA (14.0-fold) genes, and downregulation of the blaOXA-48 (5.3-fold) gene. The hvKp strain of ST86K2 demonstrated upregulation of only one gene, iroN (4.1-fold), and downregulation of 13 genes: blaSHV (2.6-fold), acrA (9.0-fold), acrB (10.0-fold), oqxB (10.1-fold), kpnE (9.9-fold), kpnF (5.0-fold), kexD (4.9-fold), kdeA (4.8-fold), iroD (4.0-fold), uge (3.2-fold), wabG (6.2-fold), treC (4.8-fold), and ureA (5.3-fold). The hybrid hvKp-MDR strain of ST23K1 showed upregulation of the acrB (3.4-fold), oqxA (2.1-fold), oqxB (2.1-fold), wabG (6.2-fold), fimH (3.2-fold), celB (8.0-fold), and ureA (8.9-fold) genes. The second hvKp-MDR strain of ST23K57 expressed upregulation of the blaTEM (4.1-fold), blaCTX-M (5.3-fold), ompK36 (2.9-fold), acrB (2.3-fold), oqxA (2.9-fold), oqxB (4.6-fold), kpnE (2.3-fold), kpnF (2.0-fold), kexD (3.2-fold), kdeA (11.7-fold), uge (3.4-fold), wabG (14.5-fold), fimH (2.9-fold), celB (3.7-fold), and ureA (3.1-fold) genes  genes .K47 demonstrated downregulation of the blaTEM (3.3-fold) and acrA (2.4-fold) genes. The MDR strain of ST147K64 was characterized by upregulation of the oqxB (2.7-fold), kpnE (7.0-fold), kpnF (4.4-fold), kdeA (2.3-fold) genes, and downregulation of the blaOXA-48 (2.1-fold) gene. The hybrid hvKp-MDR strain of ST23K1 showed upregulation of the fimH (2.1-fold) gene, and downregulation of the blaTEM (2.1-fold), kdeA (5.1-fold), and ureA (2.7-fold) genes. The second hybrid hvKp-MDR strain of ST23K57 expressed upregulation of the blaSHV (2.3-fold), blaTEM (2.4-fold), blaCTX-M (6.3-fold), acrB (3.0-fold), oqxB (3.8-fold), kpnE (2.1-fold), kdeA (8.6-fold), wabG (14.8-fold), and ureA (4.2-fold) genes, and no genes were downregulated  [(16–20%) , and sig(16–20%) .K. pneumoniae isolates collected from 62 patients were attributed to specific genetic lines, virulence, and antimicrobial resistance genotypes. Single isolates were collected from 54 patients and two isolates from eight patients. Double isolates were studied from one patient in a case of their differences in ST , K-type (Patient 35), and antimicrobial resistance genes profiles (Patients 4 and 39) (K64 carried (blaTEM+blaSHV+blaCTX-M+blaOXA-48+blaNDM) and (blaSHV+blaOXA-48) beta-lactamase genes, respectively. Two isolates obtained from the trachea of Patient 39 belonged to ST23K57 (blaTEM+blaSHV+blaCTX-M) and (blaSHV+blaCTX-M+blaOXA-48) beta-lactamase genes, respectively. Different antimicrobial resistance gene profiles of K. pneumoniae named isolates possibly indicate the evolution events in the patient’s body as described previously [Non-duplicate 70  and 39) . Two isoeviously .K. pneumoniae genetic lines common for classical K. pneumoniae (cKp) (ST395 and ST147) and hypervirulent K. pneumoniae (hvKp) (ST23 and ST86). ST395 was reported as prevalent for cKp-MDR strains in studies from Poland, France, Italy, and Russia [K. pneumoniae of ST23 and ST86 were described previously as hvKp causing bacteremia, sepsis, and liver abscess in India, France, Taiwan, and Russia [K. pneumoniae isolates of K64 commonly were recognized as cKp, but recently some isolates of K64 were described as hybrid hvKp-MDR due to acquiring the hvKp virulence plasmids [K20/K64, ST395K2, as well as hvKp ST23K1 and ST86K2 [K. pneumoniae isolates in our study were MDR with resistance to carbapenems for 45.7% isolates. Such high MDR rates were reported from China and Iran [blaCTX-M in our study was 74.2%, carbapenemase genes blaOXA-48 and blaNDM at 40.0% and 11.4%, respectively, similar to the prevalence of these genes published previously [n = 11) were attributed to ST23K1, ST86K2, ST23K57, ST65K2, and ST218K57 genetic lines. All of them carried the rmpA gene coding the major virulence-associated factor in hvKP isolates [rmpA-positive isolates in this study were hypermucoviscousity-positive, which is in agreement with a previously published report [K. pneumoniae among hypermucoviscousity-positive isolates in this study was 6/11, which is consistent with the modern trend for the appearance of hybrid hvKp-MDR genetic lines [Twenty STs and 17 K-types were identified. The most prevalent sequence types were ST395 27.1%), ST23 18.6%), ST147 15.7%), and ST86 (7.1%). The remaining 16 STs were each represented at ≤ 4.3%. The dominant STs were described previously as .7%, and .6%, ST14d Russia ,20,21,22plasmids . Moreoved ST86K2 ,27,28,29.1%, ST23and Iran ,31. The isolates , althougic lines ,34.K. pneumoniae strains belonging to prevalent STs and K-types  were selected for further comparative study of whole-genome sequences, antimicrobial resistance phenotypes, hypermucoviscosity, and altered expression levels of virulence and resistance genes in response to beta-lactams effect (AMP and CRO). It was shown that K. pneumoniae isolates belonging to ST86K2, ST23K1, and ST23K57 demonstrated hypermucoviscosity phenotype in contrast with isolates of ST395K47 and ST147K64, which confirmed the virulent phenotype of three isolates. All K. pneumoniae isolates carried blaSHV genes, including extended-spectrum beta-lactamase (ESBL) variant blaSHV-12, broad-spectrum variants blaSHV-28 and blaSHV-33, and narrow-spectrum variants blaSHV-67 and blaSHV-190. These alleles of blaSHV genes were previously described in Portugal, Turkey, China, Russia, and Spain [K. pneumoniae isolates  carried the blaTEM-1B, blaCTX-M-15, and blaOXA-1 genes. It was reported that these genes were horizontally transferred by the IncFIA-FIB-FII and IncHI2 plasmids [K57) carried the blaCTX-M-55 and blaOXA-1 genes; such gene combination was reported previously from China [K47 and ST147K64 additionally carried two carbapenemase genes, i.e., blaOXA-48 and blaNDM-1. Previously, it was reported that K. pneumoniae clinical isolates of ST395 and ST147 harbored blaNDM-5 and blaOXA-181/232 in Nepal and that ST11 harbored blaNDM-1 and blaOXA-48 in Greece [K. pneumoniae isolates belonging to the hvKp evolutionary branch, which acquired the resistance genes and became a hybrid hvKp-MDR. In our study, a hvKp-MDR isolate of ST23K1 carried simultaneously blaCTX-M-15 and blaOXA-48 genes, similar to a recently published study [K57 carried not only the blaCTX-M-55 and blaOXA-48 genes but additionally the blaNDM-1 gene. This is the first report describing K. pneumoniae of ST23K57 genetic-line-acquired cefalosporinase gene blaCTX-M-55, and two carbapenemase genes blaOXA-48 and blaNDM-1, which is particularly alarming. The incidence of high-risk clone ST383 carrying blaCTX-M-14b gene and two carbapenemase genes blaNDM-1 and blaOXA-48 combining both resistance and virulence elements was recently published [blaCTX-M-15, blaNDM, and blaOXA-181 genes [acrA, acrB, oqxA, oqxB, kpnE, kpnF, kdeA, and kexD) because of their clinical significance for K. pneumoniae beta-lactam resistance presented recently [K1 carried four efflux pump genes: acr and oqx of RND-type systems, kde of MATE-type, and kpn of SMF-type. The rest of the four strains carried five efflux pumps: acr, oqx, kde, kpn, and additionally the kex gene of the RND-type efflux system. The same efflux pump genes were detected recently in K. pneumoniae clinical isolates, which exhibited co-resistance to beta-lactams and aminoglycosides, glycopeptides, fluoroquinolones, and tetracyclines in India [Five nd Spain ,35,36. Tplasmids ,38. One n Greece . Of greaed study . Moreove81 genes . Additiorecently . In our in India . Interesin India .peg-344, iroB, iucA, plasmid-encoded rmpA, and rmpA2 and quantitative siderophore production (entB and ybtS) [K2, ST23K1, and ST23K57. In contrast, K. pneumoniae virulence genes , common for both hvKp and cKp, were detected in all five isolates. This is in agreement with recently published data [K1 and ST23K57 are characterized as hybrid hvKp-MDR. Thus, the data obtained in this study indicate the ongoing formation of hybrid K. pneumoniae on the base of the ST23 genetic line, which was already defined in the last decade [It is known that multiple biomarkers have been shown to predict hvKp isolates: nd ybtS) . In thist decade ,35,45.K. pneumoniae resistance and virulence genes at non-selective conditions in vitro and the fold change of expression levels in presence of AMP and CRO. It was shown that ESBL gene blaSHV-12 expressed ~4-fold higher in the MDR strain of ST395K47 than blaSHV-type genes coding broad-spectrum and narrow-spectrum beta-lactamases in the remaining K. pneumoniae strains. This is in agreement with previously reported data that ESBL variants of the blaSHV-12 gene expressed higher than non-ESBL variants [We estimated the basal expression levels of variants . These gblaTEM,blaCTX-M, blaOXA-48, and blaNDM and porin gene ompK36 were expressed at higher levels, with the exception of blaTEM in the hvKp-MDR strain of ST23K1. Notably, the expression levels of the beta-lactamase (blaTEM and blaCTX-M) and carbapenemase  genes were higher in cKp-MDR strains than those in hybrid hvKp-MDR strains. Possibly, the reason for this observation is the higher metabolic load in Klebsiella cells producing resistance and virulence factors simultaneously. Growing at AMP conditions for 90 min induced upregulation of blaTEM gene expression in cKp-MDR of ST147K64 (2.1-fold), as well as the blaCTX-M (5.3-fold) and ompK36 (2.8-fold) genes in hvKp-MDR of ST23K57, but downregulation of blaTEM gene expression in cKp-MDR of ST395K47 and the blaOXA-48 gene in cKp-MDR of ST147K64. In contrast, growing at CRO conditions induced upregulation of the blaCTX-M gene in hvKp-MDR of ST23K57 (6.5-fold) and downregulation of the blaTEM gene in cKp-MDR of ST395K47 (3.3-fold), in hvKp-MDR of ST23K1 (2.1-fold), and the blaOXA-48 gene in cKp-MDR of ST147K64 (2.1-fold).Beta-lactamase genes ropD, and 2–3 genes were lower than the reference. In the hvKp strain, one efflux gene expression was higher, three genes were expressed at the same level, and four genes were lower than the reference gene. In contrast, major efflux genes in hybrid hvKp-MDR strains were expressed lower than the rpoD gene, and one gene in the strain of ST23K57 was expressed on the same level as the reference. Interestingly, the previously described expression of the arcB efflux gene showed upregulation of this gene in carbapenem-resistant K. pneumoniae strains compared with non-resistant ones [Klebsiella evolutionary branches that are consistent with previous reports [K. pneumoniae genetic lines may reflect differences in bacterial surface structures in particular K-types: downregulation in the strains of K47 and K2, and upregulation in the strains of K1 and K57 in response to AMP; and upregulation in the strains of K64 and K57 in response to CRO  were present at higher levels in the cKp strains of ST395K47, ST147K64, and hvKP ST86K2 than in the hybrid hvKp-MDR strains of ST23K1 and ST23K57. In conditions containing 100 mg/L AMP, these genes were upregulated in cKp and hybrid hvKp-MDR strains (fimH 2.3-4.6-fold) and wabG in hvKp-MDR strains (6.1–14.9-fold), while downregulated in the hvKp strain . In conditions with 10 mg/L CRO, only the wabG gene was upregulated in hybrid hvKp-MDR strains (2.3-14.9-fold). The expression levels of the remaining virulence genes common for cKp and hvKp (celB and ureA), as well as common for only hvKp , were approximately equal in all studied strains at non-selective conditions. The celB gene expression at AMP medium was upregulated in hvKp-MDR strains (3.7–8.0-fold); the ureA gene expression was upregulated in the cKp strain of ST147K64 (13.9-fold) and hvKp-MDR strains of ST23K1 (9.2-fold) and ST23K57 (3.2-fold). It was detected that CRO induced upregulation of the ureA gene in a hvKp-MDR strain of ST23K57 (4.3-fold) and downregulation of this gene in a hvKp-MDR strain of ST23K1 (2.6-fold). Expression levels of virulence genes common for hvKp strains were not changed under selective pressure generated by AMP or CRO in the growth media  in a specialized Neurosurgical Hospital in Moscow, Russia. Following the requirements of the Russian Federation Bioethical Committee, each patient signed informed voluntary consent to treatment and laboratory examination. The materials used in the study did not contain the personal data of patients.K. pneumoniae isolates were collected from the respiratory system, blood, urine, cerebrospinal fluid, and wounds of 62 patients of the neuro-ICU. Bacteria identification was performed using by a Vitek-2 Compact instrument  and a MALDI-TOF Biotyper . Bacteria were grown at 37 °C with agitation on Luria-Bertani broth  and Muller-Hinton broth . Bacterial isolates were stored in 20% glycerol at minus 80 °C.Seventy n-101 and AST n-102 cards . The results were interpreted according to the European Committee on Antimicrobial Susceptibility Testing . E. coli strains ATCC 25922 and ATCC 35218 were used for quality control. Strains non-susceptible to ≥1 agent in ≥3 antimicrobial categories were identified as multi-drug resistant (MDR), according to Magiorakos et al., 2012 [Minimal inhibitory concentrations (MICs) of ampicillin (AMP), ampicillin-sulbactam (SAM), cefuroxime (CXM), cefoxitin (FOX), ceftriaxone (CRO), ceftazidime (CAZ), cefoperazone-sulbactam (CSL), cefepime (FEP), ertapenem (ETP), imipenem (IPM), tetracycline (TET), tigecycline (TGC), ciprofloxacin (CIP), chloramphenicol (CHL), gentamicin (GEN), tobramycin (TOB), amikacin (AMK), trimethoprim-sulfamethoxazole (SXT), and nitrofurantoin (NIT) were determined using a Vitek-2 instrument with VITEK-2 using AST l., 2012 .K. pneumoniae strains growing on the plates with Luria-Bertani broth  overnight at 37 °C [K. pneumoniae formed viscous strings >5 mm length using a standard bacteriological loop.The string test was used for the identification of hypermucoviscous at 37 °C . The posK. pneumoniae isolates were determined by the Multilocus Sequence Typing (MLST) scheme of Pasteur Institute  using the previously published primers [K. pneumoniae isolates was performed using specific primers for the wzy gene associated with K serotypes K1, K2, K20, and K57 [wzi gene sequencing for identification of capsular types K23, K27, K28, K31, K47, K60, K62, and K64 [Sequence types (STs) of  primers ,51. The  and K57  and by w and K64 . BacteriblaSHV, blaCTX-M, blaTEM, blaOXA-48, blaKPC, blaVIM, blaIMP, and blaNDM, class 1 and 2 integrons, and porin protein gene ompK36, as well as 8 genes associated with K. pneumoniae virulence, rmpA (hypermucoid phenotype regulator), aer (aerobactin), kfu (ferric absorption system), uge , wabG (glucosyltransferase), fimH (fimbria type I), allS and allR , were detected by PCR using specific primers as was described previously [Beta-lactamase genes eviously .http://www.generunner.net/, accessed on 1 November 2021) and in silico analysis by insilico.ehu.eus [K. pneumoniae virulence genetic determinants  and antibacterial resistance genes  were detected by RT-PCR using qPCRmix-HS SYBR  and the CFX96 Real-Time PCR system  with the following program: 95° for the 20s, 61° for 20s, 72° for 30 s, repeat 40 times. Bacterial thermolysates were used as DNA templates.Oligonucleotides for RT-PCR  were des.ehu.eus  was perfhttps://github.com/ncbi/pgap, accessed on 1 November 2021). Antimicrobial resistance genes, STs, plasmids, and restriction-modification systems were identified using the web resource of the Center for Genomic Epidemiology . Virulence genes, capsular type, genes conferring resistance to heavy metals, and efflux pumps were identified by the Institut Pasteur, Paris, France, BIGS database web-resource of .WGS was carried out on the Illumina MiSeq platform using Nextera DNA Library Preparation Kit  and MiSeq Reagent Kits v3 . The obtained single reads were collected into contigs using the SPAdes 3.9.0 software . De novo assembled genomes were annotated in the GenBank database  containing/not containing antimicrobials (100 mg/L AMP or 10 mg/L CRO) and incubated at 37 °C with agitation for 90 min. Each experiment was represented by three independent repeats. All following steps for RNA isolation were performed at 4 °C to limit RNase activity. Total RNA was isolated by phenol-chloroform extraction using kit RNA-extran , following manufacturer protocol. After isolation, RNA was treated with TURBO DNase  to remove traces of genomic DNA.proC, recA, and rpoD expression. Three technical replicates per each of the three biological samples were used for statistical validity. The relative transcript levels of antimicrobial resistance and virulence genes were calculated using the 2−ΔΔCt method [www.graphpad.com accessed on 1 November 2021). Gene expression levels of each gene at present of AMP and CRO were compared to those in conditions without antimicrobials.One µg of isolated total RNA was used for cDNA synthesis with RevertAid RT Reverse Transcription Kit . qPCR was performed using qPCRmix-HS SYBR  and the CFX96 Real-Time PCR system  with the following program: 40 cycles of 20 s at 95 °C for denaturation, 20 s at 61 °C for annealing, 30 s at 72 °C for extension and SYBR Green detection. The melting curve analysis in the temperature range from 60 °C to 94 °C, with a fluorescence estimation step of 0.2 °C, was performed to confirm the specificity of the reaction. Relative quantification of the target gene expression was normalized with reference genes t method . A heat