L. interrogans isolates from Malaysia. All six contained the larger (4.28–4.56 Mb) and smaller (0.34–0.395 Mb) chromosome typical of human pathogenic Leptospirae and 0–7 plasmids. Only 24% of the plasmid sequences could be matched to databases. We identified a chromosomal core genome of 3318 coding sequences and strain-specific accessory genomes of 49–179 coding sequences. These sequences enabled detailed genomic strain typing  and phylogenetic classification (whole-genome SNP genotyping). Even though there was some shared synteny and collinearity across the six genomes, there was evidence of major genome rearrangement, likely driven by horizontal gene transfer and homologous recombination. Mobile genetic elements were identified in all strains in highly varying numbers, including in the rfb locus, which defines serogroups and contributes to immune escape and pathogenesis. On the other hand, there was high conservation of virulence-associated genes including those relating to sialic acid, alginate, and lipid A biosynthesis. These findings suggest (i) that the antigenic variation, adaption to various host environments, and broad spectrum of virulence of L. interrogans are in part due to a high degree of genomic plasticity and (ii) that human pathogenic strains maintain a core set of genes required for virulence.The ability of Leptospirae to persist in environments and animal hosts but to cause clinically highly variable disease in humans has made leptospirosis the most common zoonotic disease. Considering the paucity of data on variation in complete genomes of human pathogenic Leptospirae, we have used a combination of Single Molecule Real-Time (SMRT) and Illumina sequencing to obtain complete genome sequences of six human clinical  Leptospira spp. colonize the proximal renal tubules of reservoir animal hosts and are excreted through urine into the external environment. Humans acquire Leptospira spp. infection through contact with body fluids  of carrier animals, such as rodents, and contaminated water and soils [Leptospira was traditionally considered to comprise approx. 20 species, but more detailed delineation by genomic techniques has recently increased this number to nearly 100 [L. interrogans is probably the most important species causing human leptospirosis worldwide. At the same time, it can infect or colonize all mammals and was reported to be the most common Leptospira species among rodents in South East Asia [Pathogenic nd soils . Thus, tnd soils . There and soils . In Maland soils . The genarly 100 . Leptosparly 100 . L. inteast Asia . Of the ast Asia .L. interrogans genome and variations therein among field isolates and serovars. Ren et al. described the first whole-genome sequence of L. interrogans, which revealed 4668 predicted genes divided between a large (4.33 Mb) and a small (359 kb) chromosome and cardinal differences from parasitic spirochetes [L.interrogans species [L. interrogans and the less pathogenic species L. borgpetersenii showed that L. interrogans possesses more signal transduction systems, transcriptional regulatory factors, and metabolic and solute transport functions than L. borgpetersenii, which may be one explanation for the remarkable ability of L. interrogans to adapt to various environments [Escherichia coli [In order to facilitate genome-based diagnostics and molecular outbreak investigations, as well as to understand the molecular evolution of phenotypic traits and genomic determinants of persistence and virulence, there have been intense efforts to elucidate the organization of the  small 35 kb chrom species . A comparonments . A subseronments . Transfehia coli .Leptospira were made in 2016. Xu et al. reported the Leptospira pangenome based on the draft genome sequences  of 102 global isolates, which revealed major genome variability across the genus as well as evidence of horizontal gene transfer and gene duplication during the evolution of pathogenic species [Leptospira species, Fouts et al. identified several features of pathogenic species, including sialic acid biosynthesis, vitamin B12 autotrophy and other metabolic features, the presence of CRISPR/Cas systems, specialized protein secretion systems, and motility and chemotaxis systems [L. interrogans isolates), Noda et al. used core genome multilocus sequence typing for genomic classification  and molecular epidemiological studies [L. interrogans serovars Copenhageni and Icterohaemorrhagiae to identify serovar-specific SNPs and indels and identified a frameshift mutation in the pic120008 gene that was specific for Icterohaemorrhagiae [Major advances in our understanding of genomic diversity in the genus  species . In a co systems . Subsequrrhagiae .Leptospira extends into the species interrogans. However, these studies were based on draft genomes and did not feature systematic comparisons of genome organization and rearrangement based on closed genomes. We have, therefore, combined Single Molecule Real-Time (SMRT) and Illumina sequencing to obtain whole-genome sequences of six clinical L. interrogans isolates that were collected from a geographically defined area , and defined their cardinal shared and distinct features regarding genomic variability and potentially pathophysiologically relevant genes. Furthermore, the obtained sequences allowed an evaluation of different in silico classification schemes  for taxonomic classification of the species L. interrogans.Taken together, these studies suggested that the high degree of inter-species genomic variability in the genus L. interrogans in moist environments and the broad spectrum of clinical manifestations and severity and identify 1530 as the strain associated with the mildest disease severity.Clinical information, including risk factors for infection, laboratory findings, and clinical severity, is summarized in L. interrogans strains were assigned to the traditional serogroups by microagglutination test (MAT), using a panel of 43 polyclonal rabbit anti-Leptospira reference sera, representing 24 pathogenic and two saprophytic serogroups  to 4.91 (782) Mb with about 3600 to 4300 CDS, but the differences were particularly due to the high number of plasmids in strain 782 and 1489 and their absence in strain 898. All strains had two 23S, two 16S, and one 5S rRNA genes. As previously described , the rRNL. interrogans. Compared with the reference genome L. interrogans ATCC 43642T [pntA and pfkB genes were identified in Langkawi and 1530, respectively  species typing A and DNAC 43642T , strain C 43642T . Resultsectively .L. interrogans strains, comprising the 6 and 15 Malaysian strains whose complete and draft genomes, respectively . Core genome alignment using Parsnp was applied to these 29 L. interrogans strains. It created 2599 clusters, with an average of 16 maximal unique matches (MUMs) per cluster (average cluster length 1327 bp), corresponding to a 75.5% coverage of the reference genome, i.e., serovar Lai, strain 56601. A phylogenetic tree was generated using the identified core genome SNPs (n = 2599) and the maximum likelihood method  were grouped in the same cluster in the whole genome SNP tree. We also noted that strain 898 is closely related to serovar Icterohaemorrhagiae (length difference 0.00%). Based on Malaysian epidemiological data, this is quite plausible as Icterohaemorrhagiae is a common circulating serovar in Malaysia [L. interrogans in a high endemicity country like Malaysia.We then performed whole-genome SNP genotyping to analyze the inter-strain relationships of the 29 d method B, which Malaysia . AlthougIn order to visualize the overall genome organization and to uncover potential genome rearrangements across strains, conserved syntenic regions were visualized using Mauve genome aligner . Large crfb locus encodes the O-antigen, which is the basis for Leptospira serovar identification and also contributes to pathogenicity [rfb loci of the six strains against Fiocruz L1-130 as the reference genome  but not within the three Icterohaemorrhagiae strains  and therefore most presumably inactive.The genicity . Serovargenicity . We compe genome . The cru strains . In all Phages and CRISPR systems. In silico phage finding suggested that the six strains contained between 1 and 10 phages . Pathogenic Leptospirae possess more than 20 types of IS in their genome and IS are considered among the driving forces of leptospiral genome evolution and diversification [Lin1 followed by ISLin2, both being located on chromosome I.fication . A detaikdsB1), which divided the strains into two groups based on their similarity to serovar Copenhageni (Fiocruz L1-130) and the Icterohaemorrhagiae serogroup: strain Langkawi, strain 898, and strain 1530, all of which belong to serogroup Icterohaemorrhagiae, had high similarity, whereas strains 782, 1489, and 1548 had low similarity. The sequences of the other 12 enzymes involved in lipid A biosynthesis were 99–100% identical to each other  in all targets except UDP-N-acetylglucosamine diphosphorylase.We compared amino acid sequences of genes involved in synthesis of lipid A, a component of the LPS layer that has different acyl chains and phosphorylation sites that can abrogate endotoxinogenicity . The sixch other . Althougch other . The degL. interrogans genomes contained up to 10 copies of scattered algl  in chromosome I homologous to Pseudomonas aeruginosa (P. aeruginosa) PAO1 (ranging 90–99%), suggesting that these strains could produce alginate-containing biofilms. In order to test how extensive the homology was across the entire pathway, we queried for homologies with all P. aeruginosa proteins potentially involved in alginate biosynthesis and transport, as well as the factors involved in phenotypic/regulatory switching in this pathway , and high conservation only for one regulatory (AlgB) and one phenotypic switching (MucD) factor [P. aeruginosa in terms of extracellular export and phenotypic regulation  factor . Thus, eerrogans . All of In order to identify additional potential virulence factors, we used the Virulence Factor Database (VFDB) as reference under the assumption that a virulence factor that has been validated in other pathogenic bacteria is likely relevant to pathogenic leptospires. Among other potential virulence factors, this analysis identified enzymes involved in membrane signaling, tissue degradation, complement inactivation, oxidative stress responses, antiphagocytic capsule, efflux pump, chemotaxin proteins, and type II and III secretion systems .A search for CDS that are potentially associated with antimicrobial resistance did not reveal significant homologies with any plasmid-encoded or chromosomal resistance genes known from other bacteria.L. interrogans strains from Malaysia, a country with high endemicity of this pathogen, enabling the mapping of a definitive genome structure and subsequent detailed genome-wide comparisons.Considering the persisting challenges in taxonomic classification, diagnosis, and genotype–phenotype correlations in pathogenic Leptospirae, we sequenced and acquired the complete genome sequences of six rfb locus, which is the key antigen in host immunity to leptospirosis and the classic antigen used for serotyping. The number of plasmids varied highly among the six strains. Intriguingly, only a minority of plasmid-encoded sequences could be matched with known sequences, and it is tempting to speculate that some of the remaining sequences contribute to adaptation and virulence by virtue of potentially novel functions.We found, on one hand, considerable shared synteny in gene organization and contents, but also evidence of massive genomic rearrangement and plasticity, as well as greatly varying strain-specific and overlapping (shared by 2–5 strains) accessory genomes. These seemingly contradictory findings are best explained by the need of the pathogen to maintain a core genome that mediates the functions for persistence and transmission in a common “envirome”, but at the same time to provide enough responsiveness in the genome to allow for adaptation in the face of environmental and immune-mediated pressure. The potential for genomic flexibility is manifested in these strains primarily by the wide occurrence of mobile elements, i.e., plasmids, insertion sequences, and prophages, which were found in all six strains but varied highly in number and distribution throughout the genome. It has been suggested that such mobile elements support ongoing genome plasticity by homologous recombination and horizontal gene transfer . It is wLeptospira genomes [Previous studies have shown that prophages occur in  genomes  and that genomes . Prophag genomes . In our  genomes .L. interrogans virulence, including 100% amino acid sequence identity in all 13 factors that had been defined by Murray et al. as experimentally validated in acute infection [L. interrogans encodes a much broader range of virulence factors than previously thought, which opens up exciting opportunities to identify additional determinants of clinical severity in humans as well as potential treatment targets. Interestingly, we found strong homologies to the antiphagocytic cap8EFG protein from S. aureus in the O-antigen locus [L. interrogans serogroup Bataviae a sequence homologous to complement-inactivating protein NeuBC from Streptococcus agalactiae is found in this locus, further underscoring the importance of plasticity within this locus [Neisseria gonorrhea. Its presence in four copies in all strains indicates that it is one of the essential defensive strategies used by L. interrogans [We found considerable conservation of loci previously implicated in nfection . In addien locus , whereasis locus . Anothererrogans , for ins−52) with T2SS from other pathogenic bacteria in all six strains, i.e., GspD and GspE from Shigella dysenteriae and XcpS from P. aeruginosa, the latter of which is required for the translocation of a variety of toxins and enzymes across the outer membrane into extracellular fluid [Chlamydia trachomatis, in all six L. interrogans strains with a conserved locus tag. In C. trachomatis, CdsN is an ATPase that catalyzes the unfolding of proteins and the secretion of effector proteins through the injectisome and interacts with a putative chaperone, Cpn0706, and the putative plug and effector protein. However, we did not find homologies with Cpn0706 or CopN in L. interrogans, and the function of CdsN as a T3SS in L. interrogans therefore remains doubtful [As opposed to some Gram-negative bacteria, which possess six or more types of secretion systems, only the Type 1 (T1SS) and Type 2 (T2SS) secretion systems have been reported in Leptospirae . Althougar fluid ,36. It idoubtful .L. interrogans; however, these findings may change as more isolates are examined [Consistent with the nearly universal susceptibility of Leptospirae to penicillins, we did not detect any plasmid-encoded antibiotic resistance genes such as beta-lactamases. Likewise, there were no acquired mutations associated with antimicrobial resistance in other bacterial pathogens. These results support clinical experience that antibiotic resistance is still not an emerging problem in examined .L. interrogans isolates 782, 898, 1489, 1548, and 1530 were isolated from five patients admitted to HTAR with a clinical diagnosis of leptospirosis. Clinical and laboratory data of the five patients were recorded. One isolate originating from Langkawi, Malaysia [In total, 2000 blood samples from patients suspected of leptospirosis admitted to Hospital Tuanku Ampuan Rahimah (HTAR), Klang, Selangor , were collected from 1 January 2014 until 31 December 2015. Samples were cultured in modified Ellinghausen–McCullough–Johnson–Harris (EMJH) media and incubated at 30 °C for four weeks, as described . L. inteMalaysia , was purL. interrogans culture in 30 mL of EMJH medium was centrifuged and the pellet was resuspended in 180 μL Buffer ATL . Genomic DNA extraction from the isolates was performed using the QIAamp DNA Mini Kit. The extracted DNA was stored at −70 °C until sequencing.For DNA extraction, a 7–10-day-old The SMRTbell™ template library was prepared according to the instructions from Pacific Biosciences  following the Procedure & Checklist—Greater Than 10 kb Template Preparation. To prepare 15 kb libraries, 8 µg genomic DNA was sheared using g-tubes™ from Covaris . DNA was end-repaired and ligated overnight to hairpin adapters applying components from the DNA/Polymerase Binding Kit P6 (Pacific Biosciences). BluePippin™ Size-Selection  to greater than 4 kb was performed according to the manufacturer’s instructions. Conditions for annealing of sequencing primers and binding of polymerase to purified SMRTbell™ template were assessed with the Calculator in RS Remote (Pacific Biosciences). SMRT sequencing was carried out on the PacBio RSII (Pacific Biosciences) taking one 240-min movie for one to three SMRT cells per isolate using P6 Chemistry.® Ultra™ DNA Library Prep Kit for Illumina® with 550 bp as insert size. Sequencing using the MiSeq Personal Sequencer  was done to 250 cycles in both directions. The generated sequencing reads were de novo assembled with the VELVET tool version 1.2.10. (www.ebi.ac.uk/~zerbino/velvet/ (accessed on 29 March 2017)) [For short reads, libraries for whole genome sequencing were prepared with the NEBNexth 2017)) .dnaA or the respective plasmid replication gene as the first gene. Error correction was performed by mapping of Illumina MiSeq data onto finished genomes using BWA 1.1.4 (http://bio-bwa.sourceforge.net/ (accessed on 29 March 2017)) [http://varscan.sourceforge.net/ (accessed on 29 March 2017)) [https://kbase.us/applist/apps/ProkkaAnnotation.net/ (accessed on 29 March 2017)) [https://www.ncbi.nlm.nih.gov/genome/annotation_prok/ (accessed on 29 March 2017)) under Accession Numbers CP043876-CP043901 and VWNG00000000, respectively.SMRT Cell data were assembled using the “RS_HGAP_Assembly.3” protocol included in SMRT Portal version 2.3.0 using default parameters. The assemblies revealed circular chromosomes and plasmids. All replicons were circularized, and particularly the artificial redundancies at the ends of the contigs were removed and adjusted to h 2017)) , with thh 2017)) . A conseh 2017)) . The genL. interrogans were obtained from NCBI RefSeq and used as query genomes. In addition, we obtained 15 previously unsequenced Malaysian strains from Leptospirosis Reference Centre, Amsterdam, and prepared draft genomes by Illumina sequencing.Seven additional complete genomes of Typing with polyclonal reference sera  and monoclonal antibodies to identify serogroups and serovars was performed by the WHO Leptospirosis Reference Centre, Amsterdam, as described before .The CAAT protocol was described in . First, http://ggdc.dsmz.de/ (accessed on 13 May 2017)), with 70% similarity as the gold standard threshold for species boundaries. The results indicated that the G + C content, if computed from genome sequences, varied no more than 1% within species. The sequence of the reference strain, L. interrogans ATCC 43642T, was downloaded from https://www.ncbi.nlm.nih.gov/assembly/GCF_900156205.1 (accessed on 13 May 2017).Genome-to-Genome Distance Calculator (GGDC) is an in silico tool for genome-to-genome comparison that mimicks conventional DDH . The DNAhttps://tygs.dsmz.de (accessed on 13 May 2017)) was used to infer a phylogenetic tree with FastME 2.1.6.1 from GBDP distances calculated from genome sequences. The branch lengths are scaled in terms of GBDP distance formula d5. The numbers above the branches are GBDP pseudo-bootstrap support values > 60% from 100 replicates, with an average branch support of 61.4%. The tree was rooted at the midpoint analyses [The Type (Strain) Genome Server  were extracted from all 29 genomes, and sequence types (STs) assigned using the MLST website (http://leptospira.mlst. net/ (accessed on 20 April 2017)) [The nucleotide sequences of seven MLST housekeeping genes (l 2017)) .https://github.com/marbl/parsnp (accessed on 13 May 2017)) [L. interrogans serovar Lai strain 56601 (RefSeq NC_004342.2). Within ParSNP, FastTree2 was used to compute a SNP tree, which was computed using the maximum likelihood algorithm with 1000 permutations. Output was produced as multi-alignments (XMFA), variants (VCF), core genome phylogeny (Newick), and Gingr input format (GGR). The Newick files were later exported into MEGA 7 [Genome-wide single nucleotide polymorphism (SNP) patterns were analyzed with ParSNP v1.2 (y 2017)) , a core o MEGA 7 , for obtThe following general parameters of five complete genomes and one draft genome were documented: size of chromosome I and II; number of contigs in each genome; and number of tmRNA, rRNA, tRNA, repeat regions, genes, coding sequences (CDS), signal peptides, and miscellaneous RNA.https://www.bioinf.uni-leipzig.de/Software/proteinortho/ (accessed on 23 May 2017)) [http://jvenn.toulouse.inra.fr (accessed on 17 August 2021)) [Clusters of orthologous proteins were generated using ProteinOrtho V5.16b (y 2017)) . The cort 2021)) .https://www.mybiosoftware.com/mauve-2-3-1-multiple-genome-alignments.html (accessed on 25 May 2017)) [An analysis for genome synteny applying LCB was performed using Mauve 2.3.1 2.4 (y 2017)) .http://eggnog5.embl.de (accessed on 25 September 2017)) [Contigs that did not belong to chromosome I and II were considered plasmids. The fasta files of the contigs were queried in Blastn for identification of the plasmids. Orthologous Group (OG)-based functional annotation of CDS predicted in each plasmid was performed using eggNOG 5.0 (r 2017)) .http://phaster.ca (accessed on 25 September 2017)) [Genomes were queried for the presence of phages using PHASTER (PHAge Search Tool Enhanced Release) at (r 2017)) .https://crisprcas.i2bc.paris-saclay.fr/ (accessed on 25 September 2017)) [Genome sequences were queried for Clustered Regularly Interspaced Short Pallindromic Repeats (CRISPR) elements using CRISPRCasFinder (r 2017)) . Spacersr 2017)) .https://isfinder.biotoul.fr/ (accessed on 25 May 2018)) [rfb locus were queried by Blastp search [Genome sequences were compared against IS database using ISFinder (y 2018)) . Only hip search .https://cge.cbs.dtu.dk/services/ResFinder/ (accessed on 27 May 2018)) [The genomes were queried for chromosomal mutations and acquired genes associated with antimicrobial resistance genes using ResFinder 3.2 (y 2018)) .rfb locus) of all six strains was compared using EasyFig [L. interrogans serovar Copenhageni strain Fiocruz L1-130 using Blastp search (https://blast.ncbi.nlm.nih.gov (accessed on 30 May 2018)) [The LPS biosynthetic system ) . Locus thttp://www.mgc.ac.cn (accessed on 15 September 2021)) [−52, which represents the highest score of alignment, i.e., ≥200, were considered significant.Protein sequences of each genome were queried against 2602 sequences of proteins associated with experimentally verified virulence factors from other bacteria in (VFDB) (P. aeruginosa PAO1 reference strain were compared against the six L. interrogans genomes using Blastp search [Proteins associated with structural biosynthesis, regulation, and genotypic switching relating to alginate biosynthesis in the p search .Leptospira interrogans is a highly dynamic pathogen that can survive in multiple hosts and environments and causes a broad spectrum of clinical severity in humans. Our study underscores the variability and plasticity of its genome, but also highlights conserved features that may be critical for pathogenesis in humans. Future studies should aim to unravel the relative contributions of the conserved and adaptive features of this pathogen to outbreak dynamics and pathogenicity.


Helicobacter pylori, a highly diverse organism is reported to be associated with several gastrointestinal diseases like, gastritis, gastric cancer (GC), peptic ulcer, duodenal ulcer (DU) etc. A novel similarity score (Sm)-based comparative analysis with GIs of 50 H. pylori strains revealed clear idea of the various factors which promote disease progression. Two putative pathogenic GIs in some of the H. pylori strains were identified. One GI, having a putative labile enterotoxin and other dynamin-like proteins (DLPs), is predicted to increase the release of toxin by membrane vesicular formation. Another island contains a virulence-associated protein D (vapD) which is a component of a type-II toxin–antitoxin system (TAs), leads to enhance the severity of the H. pylori infection. Besides the well-known virulence factors like Cytotoxin-associated gene A (CagA) and vacA, several GIs have been identified which showed to have direct or indirect impact on H. pylori clinical outcomes. One such GI, containing lipopolysaccharide (LPS) biosynthesis genes was revealed to be directly connected with disease development by inhibiting the immune response. Another collagenase-containing GI worsens ulcers by slowing down the healing process. GI consisted of fliD operon was found to be connected to flagellar assembly and biofilm production. By residing in biofilms, bacteria can avoid antibiotic therapy, resulting in chronic infection. Along with well-studied CagA and vacuolating toxin A (vacA) virulent genes, it is equally important to study these identified virulence factors for better understanding H. pylori-induced disease prognosis.Bacterial virulence factors are often located in their genomic islands (GIs).  Helicobacter pylori, formerly known as Campylobacter pylori, is a Gram-negative, microaerophilic bacteria that is part of the gastric microbiota in over 50% of population worldwide. The International Agency for Research on Cancer (IARC) classified it as a Class I carcinogen in 1994, nearly 20 years after its discovery, stating that people having H. pylori infection have six-times higher risk of developing gastric cancer (GC) than those who do not [H. pylori in stomach is commonly acquired in early age, mostly by person-to-person transmission through fluidic or aerosolic transfer mediated by the feco-oral route [H. pylori infection leads to a variety of upper gastrointestinal disorders, such as chronic gastritis, peptic ulcer disease (PUD), gastric mucosa-associated lymphoid tissue (MALT) lymphoma, and GC [H. pylori were found to have different level of virulence, while some of the strains were found to be non-virulent. So, to understand the disease prognosis in a better way and to develop new therapeutics to fight against this bacterium, it is important to study H. pylori at the strain level. Strain-specific virulence factors are generally located in the Genomic Island (GI) region of particular pathogenic strains. Large genomic regions consisting of multiple horizontally transferred genes (HTGs) are collectively termed as GIs [strains of Escherichia coli. This identified virulence factor was not present in beneficial strains of E. coli. This cluster of genes was referred to as pathogenicity islands (PAIs). It has been proposed that possible vaccine candidates are located within PAIs [al route . H. pylo, and GC . Colonizd as GIs . These ad as GIs . In 1990hin PAIs ,11.H. pylori infection is a leading cause of GC, the majority of people who carry the bacteria in their stomachs never get diagnosed with GC. It was also found that some of the normal individuals showed to have higher abundance of H. pylori. They neither showed any symptoms nor had any discomfort. H. pylori appeared to be the part of normal microflora for these samples. So the role of H. pylori in GC prognosis is still unclear. H. pylori is a highly diverse organism. Studying H. pylori at strain level may shed light to understand the disease prognosis. In the present study, the GIs were thoroughly studied in 50 H. pylori strains in order to find strain-specific novel virulent factors and other Islands. The comparative study with the GIs among 50 H. pylori strains identified GIs with virulence-associated genes in addition to the known GIs. Further characterization of the newly detected islands identified the possible function of these GIs. Several other factors were also identified which were found to be associated either directly or indirectly with the H. pylori-induced disease prognosis.Despite the fact that H. pylori were downloaded from ftp://ftp.ncbi.nlm.nih.gov/. All of these strains had common hosts i.e. humans. Geographical distribution and their disease association are listed in Supplementary Tables S1 and S2. Design-Island-II , a de novo algorithm for GI predication was run on 50 H. pylori strains to predict the statistically significant GIs. Islands that were ≥10 kb in length were annotated as GIs. All HTGs within these GIs were identified from the gene annotation data using an in-house perl script. For comparison of the GIs among different strains, we used cd-hit suite (http://weizhong-lab.ucsd.edu/cdhit-web-server/) to assign unique Cluster numbers (CLS) to the HTGs. Sequences with 90% identity were given identical cluster (CLS) numbers.Fifty completely sequenced strains of H. pylori strains, we introduced a similarity score (Sm):To identify the similarity of a particular GI among different Design-Island-II.Sm calculates the number of common genes present in the two GIs, divided by the number of total genes present in the two GIs. We calculated Sm score for all the identified GIs in 50 H. pylori strains. Sm value-based heat maps were generated for all GIs to get an idea about the similarities or dissimilarities among the 50 H. pylori strains. Hierarchical clustering on the Sm score and heat maps were generated for all GIs using R packages (gplots and heatmap.2).where, A and B correspond to the number of genes within the given GIs as identified by Sm ≥ 50% was used to annotate a GI as shared between two strains. GIs present in all 50 strains were classified as core GIs. These were named as Core_1, Core_2, etc. Accessory GIs were defined as GIs found in 2–49 H. pylori strains and termed as Acc_1, Acc_2, Acc_3 and so on. The presence and absence of GIs across all the strains were denoted as 1 and 0. Kendall’s Tau correlation coefficient was calculated among all 50 H. pylori strains considering the presence or absence of accessory islands.A cut-off value of https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and psi blast (NCBI) against PDB database with default parameters until convergence. The query sequences were searched against CDD v3.19 - 58235 PSSMs database, using 0.010 as expected value. To understand the conservation of the homolog in the organisms and their evolutionary relationships, multiple sequence alignment and phylogenetic analysis were carried out using Molecular Evolutionary Genetic Analysis (MEGA) [A large number of genes within many accessory islands were annotated as hypothetical proteins. Functional characterization of these unknown proteins was performed using a conserved domain search (s (MEGA) .The probable biological role of the uncharacterized proteins is predicted by studying the protein–protein interaction using STRINGv10 . Three-dProtein–peptide interactions are essential in various biological processes involving signaling, cellular localization etc. Twenty-six probable complexes were generated using a protein–protein docking server ClusPro . The comH. pylori strains to identify the GIs and their locations in the genome. The length of the predicted GIs were compared with that of the GIs predicted by Design-Island-II. If at least 50% of the length of GI, predicted by any other tool was overlapped with our predicted GIs, it was determined as detected by both methods.To validate the identified GIs with other GI prediction tools, sequence composition-based approaches like, PredictBias , ZislandDesign-Island-II, a modified version of Design-Island [H. pylori strains. A total of 41 GIs with ≥10 kb length were included for the comparative analysis of 50 strains. The H. pylori 35A strain had the highest number of GIs (36 GIs), followed by two Fukui strains (H. pylori F16 and H. pylori F30) and H. pylori J99, both of which had 35 GIs. The Malaysian strains H. pylori UM037 and H. pylori UM066 had the lowest number of predicted GIs (25 GIs)  were quite different from each other and other geographical strains. On the contrary, the strains from Japan, Korea and France showed ≥0.80 correlation among themselves. South African strains H. pylori 908, H. pylori 2017 and H. pylori 2018 were almost identical in terms of shared GIs (n-Island  was used(25 GIs) A. Heat mared GIs B.H. pylori strains. Among these, seven GIs were present in all 50 strains and were termed as core GI. Three core GIs (Core_1 to Core_3) were found to have Sm ≥ 80%. The rest four core GIs (Core_4 to Core_7) had Sm ≤ 80%. Thirty-four accessory GIs  were present in 2–49 H. pylori strains with (Sm ≤ 80%)  C. The plH. pylori strains with an average mS = 83.1%. The heat map gave a lucid pictorial illustration of the similarity of Core_3 GI among strains . The length of Core_3 was ∼17 kb and 14 HTGs were present on it. This gene cluster was the entire nuo operon (NADH: ubiquinone oxidoreductase). This operon contains essential genes with unique genomic properties, such as different oligonucleotide frequencies and slightly greater GC% than tissue-specific genes differentiate this region from rest of the genome [H. pylori strains have three mutations in NADH-quinone oxidoreductase subunit D (NuoD) at G398S, F404S and V407M and one in NADH-quinone oxidoreductase subunit B (NuoB) at T27A. Despite the fact that this GI contains essential genes, it can be classified as an antibiotic-resistant island. The synteny of 14 genes in this GI revealed a framework in which homologous gene conservation and gene order were observed .Core_3 was found to be present in all e genome ,32. ThusvacA) gene along with another toxin flippase-type exporter (mviN), iron (III) dicitrate ABC transporter genes fecD and fecE, short-chain oxidoreductase (vdlC) in almost all. VacA was deleted in five H. pylori strains like, H. pylori Shi169, H. pylori UM037, H. pylori Rif1, H. pylori Rif2, H. pylori SouthAfrica7. In H. pylori 52 and H. pylori BM012S vacA was annotated as non-coding. Synteny of this GI revealed that it differed between strains depending on gene content  with average Sm score of 77%. The hierarchical clustering based on the Sm grouped the samples in two major clusters . This GI is associated with virulence as well as iron transport. H. pylori Rif1, H. pylori Rif2 strains lacked vacA gene, though in the other vacA lacking strains, vacA paralogs were present on another accessory GI, Acc_23.Core_4, a ∼17 kb long GI was detected to contain vacuolating toxin A , Venezuela strain H. pylori V225, Fukui strain H. pylori F57 showed distinct gene arrangements (The urease gene cluster (Core_5), which was adopted horizontally, became one of the most important genetic elements for the survival of e enzyme ,34. The clusters A. Three ngements B. The efH. pylori infection. This GI was present in all strains, whether intact, fragmented or missing in part. However, a U32 family peptidase was present in all strains. Other important HTGs of this GI were a DNA-binding response regulator (racR), peptide chain release factor RF2 (prfB), flagellar biosynthetic protein FliR (fliR), fructose-bisphosphate aldolase (fbaB), etc. The average Sm, calculated on the basis of gene content was found to be 64.5%. Thirty-six strains showed >80% similarity and formed the main cluster in the hierarchical clustering . U32 family peptidase present in all the strains is actually a collagenase. Gastric epithelium extracellular matrix is mostly composed of collagen type I and III. Type I collagen synthesis is increased in the areas surrounding gastric ulcers. Thus, ulcer healing process is promoted [H. pylori inhibits the healing process, thus increase the chronicity of the ulceration. Synteny of this GI showed all the 50 strains have this virulence factor, but the difference in the gene content of the adjacent region led to formation of distinct clusters .Core_6, a 13-kb long GI, was identified with genes that promote the virulence of promoted . HoweverfliD operon along with some other HTGs, like, DNA-damage induced multidrug efflux protein (dinF), N-carbamoylputrescine amidase (cpa), 2′,3′-cyclic-nucleotide 2′-phosphodiesterase (ymdA) etc. This GI was ∼12 kb long and was present in all strains. The fliD operon not only promotes H. pylori colonization in the human stomach by forming flagella, but it also facilitates biofilm formation [H. pylori strains; with 75% of average Sm score . The difference in adjacent gene cluster led to the formation of two major groups . Core_7 is involved in H. pylori colonization and biofilm formation, which contributes to the disease’s chronicity. As fliD is present in all strains and crucial for H. pylori survival, it can be a potential target for new drug or vaccine development.Core_7 was found to contain ormation . The bacormation . This oplpxB), hof-family outer membrane protein , CDP-diacylglycerol diphosphatase (cdh) etc. This GI was found in 41 strains, with an average Sm of 55.3%. The hierarchical clustering in the heat map divided the strains into two major clusters  identified that ∼80% of the strains have 70–100% of resemblance among them . It was ∼40 kb in length and consisted of 32 genes. Three strains  showed ∼90% similarity among themselves, however, differed from the other strains. These three strains, reportedly associated with duodenal ulcer (DU), belong to the same geographical location i.e. Africa. While five Cag negative strains H. pylori Aklavik 86, H. pylori Aklavik 117, H. pylori B38, H. pylori SA7 and H. pylori SA20 were clustered together. Hierarchical clustering based on the Sm, generated from gene content showed three distinct clusters. Forty-five strains Cag positive strains were either associated with a specific disease or isolated from patients. Four Fukui strains , two Australian (H. pylori BM012A and H. pylori BM012S), two German (H. pylori Rif1 and H. pylori Rif2) and two Korean (H. pylori 51 and H. pylori 52) strains showed to have identical gene content among themselves, suggesting an intraspecies horizontal transfer of CagPAI within a particular geographical location. Synteny of this island showed three representatives from three distinct clusters observed in the heat map . Gene composition analysis of the cagPAI suggested additive or reductive evolution in some of the H. pylori strains.Cag is an exhaustively studied PAI in H. pylori strains. The GI was ∼15 kb long and consisted of 11 HTGs. Diaminopimelate decarboxylase (lysA), chorismate mutase, amidase (amiE), para-aminobenzoate synthase glutamine amidotransferase (pabB) were some of the genes linked to the lysine biosynthetic process. The similarity among the strains was mS = 69%. Hierarchical clustering identified three distinct clusters. The gene content of Acc_14 in H. pylori 51, H. pylori 83 and H. pylori Lithuania 75 were quite different from others . The functional relevance of this particular GI came up with an interesting finding. Previous reports suggested consumption of a lot of salt in the diet has been linked to an elevated risk of stomach cancer [CagA), amiE, lysA, lgE2, and other proteins were found to have increased expression in bacteria cultivated in high-salt conditions [amiE, lysA enhance the risk of GC in people who take a high-salt diet. The following strains H. pylori 35A (HMPREF4655_20533), 51 (KHP_0287), H. pylori 908 (hp908_0304), H. pylori 2017 (hp2017_0297), H. pylori B8 (HPB8_1272), H. pylori B38 (HELPY_0296), H. pylori F30 (HPF30_1004), H. pylori F32 (HPF32_0300), H. pylori F57 (HPF57_0344), H. pylori J99 (jhp0275), H. pylori OK113 (HPOK113_0299), H. pylori OK310 (HPOK310_0296), H. pylori P12 (HPP12_0289), H. pylori SA20 (HPSA20_0322), and H. pylori V225D (HPV225_0308) have lysA and amiE, which thus increase the chance of GC prognosis. However, lysA and amiE were absent from H. pylori Cuz20 and H. pylori Aklavik86, respectively .Another biosynthetic island (Acc_14) was identified in 33 h cancer . Cytotoxnditions . So, thiH. pylori strains, spanning 12.6 kb in length and including 10 HTGs. Two genes found on this GI were primarily linked with the citric acid cycle: 3-oxoacid CoA-transferase A and 3-oxoacid CoA-transferase B . Other proteins found on it include acetone carboxylase subunit γ (acxC), hydantoin utilization protein A (hyuA), and acetyl-CoA C-acetyltransferase (thiA), short-chain fatty acids transporter (atoE), polysaccharide biosynthesis protein (wlaX) etc. On the basis of the gene content the average Sm was calculated to be 53.8%. All of the studied strains had scoA, scoB and acxC present on Acc_20. Variations in the neighboring gene content led to formation of four separate clusters among the strains .Acc_20 was discovered in 48 H. pylori 908, H. pylori 2017 and H. pylori 2018, isolated from DU patients. H. pylori Puno135 and H. pylori F32 were isolated from gastritis and GC patients, respectively. Synteny study of these strains differed from other H. pylori strains -lysidine synthase (tilS). TilS co-occurred and co-expressed with d-alanine-d-alanine ligase (ddl) and tRNA-dihydrouridine synthase (dus) .The binding pocket, where it binds with its ligands, was made up of 26 amino acids. Fifty percent residues of this pocket were found to be conserved among various closely related bacteria  consisted of 11 hypothetical proteins with a labile enterotoxin output A. This labile enterotoxin was found only in five pathogenic strains, namely  strains A,B, sugg strains C. Apart d Vibrio A. Proteise (dus) B. Ddl wabacteria C. The hoDesign-Island-II identified a PAI (Acc_33), consisted of a Toxin–Antitoxin system (TAs) in 29 of the studied strains. This GI contains a vapD, Cobalt-zinc-cadmium resistance protein, cation system protein, and six proteins with unknown function. These hypothetical proteins were found to be either dynamin or DUF3240 family protein. VapD is a toxin with purine-specific endoribonuclease activity, whereas DUF3240 family protein, with unknown function, binds to its own regulatory region. The toxin and antitoxin were found to be present on the GI of all strains; however, the neighboring genes within the GIs were missing in 26 strains, except H. pylori F16, H. pylori F30 and H. pylori F32 . One GI (Acc_25) was predicted by all five methods, while thirteen GIs were predicted by three methods .dictBias , ZislanddictBias  and MSGIDesign-Island-II. Among these, four GIs  contained ribosomal proteins along with some HTGs of miscellaneous functions. Many highly expressed genes, such as ribosomal proteins, diverge from the genetic background in terms of composition. As a result, despite the fact that they were essential genes, they were identified as GIs. Several studies have proposed that GI-containing ribosomal proteins be eliminated since they are false positives in terms of identification [Eleven accessory GIs were identified only by fication ,30.H. pylori taking led to identify some new statistically significant GIs in addition to the known GIs. Characterization of the detected GIs gave an idea of their impact in the H. pylori colonization, infection and disease prognosis. The nuo operon encoding NADH: ubiquinone oxidoreductase or complex I was found as a GI in all 50 strains. Complex I, present in inner mitochondrial membrane, have multisubunits and are responsible for catalyzing the first step of cellular respiration. Essential genes serve as lucrative targets of drug development. The standard H. pylori eradication therapy is done using PPIs along with antibiotics like, amoxicillin, metronidazole, clarithromycin. Benzimidazoles (BIs) were found to have potent anti H. pylori effect in metronidazole- or clarithromycin-resistant strains. However, three missense mutations at amino acid position G398S, F404S, V407M in NuoD and one point mutation at T27A in NuoB led to the evolution of BI-resistant strains [Comparative analysis among 50 strains of  strains .H. pylori. This multimeric enzyme, made up of 12 UreA and UreB heterodimers, catalyzes the hydrolysis of urea to create CO2 and NH3. It serves as a buffer against the acidity of the surrounding environment [2 and NH3 diffuse to the periplasmic region where α-carbonic anhydrase converts CO2 into the bicarbonate (HCO3−) and H+. The HCO3− maintains the periplasmic pH close to 6.1, assisting H. pylori to avoid acidic pH [+ ions, responsible for imparting acidity is also taken up by NH3 in the periplasmic region, another way of acid acclimation [H. pylori combats the deadly effect of the acidic pH. CagPAI is a well-studied virulence factor in H. pylori [cagA an onco-protein which gets transferred to the epithelial cells through T4SS. VacA is known to form vacuole, facilitates H. pylori colonize and a pore-forming toxin. The recruitment of inflammatory cells by VacA promotes ulceration. VacA mutant strains were found to produce stomach ulcers less likey than wildtype strains in gerbils [H. pylori in the human stomach mucosa with the help of urease gene cluster containing GI. Thus these three GIs play significant role in the development of H. pylori infection-mediated illnesses.The effective colonization by the bacteria in the host is the first stage towards conferring pathogenicity. The urease gene cluster is necessary not only for establishing colonization but also for maintaining infection of ironment . CO2 andcidic pH . The H+ limation . As a re. pylori . The pre. pylori . CagPAI  gerbils . CagA, aH. pylori damages the insulating mucosal barrier of the stomach and duodenum, allowing acid to penetrate the delicate lining beneath. The acid and germs both damage the lining, resulting in a sore or ulcer. The rapid regeneration of the epithelium layer is not the only mechanism for healing injury; fibrillar collagens of types I and III have been reported to be overexpressed [After the successful colonization xpressed . These sacxC, a subunit of acxABC responsible for acetone carboxylation. While H. pylori lacks succinate dehydrogenase, a key enzyme in Krebs cycle, the SCOT complex acts as an alternative for energy metabolism. On the other hand acxABC promotes acetone utilization. Thus, these two are beneficial for H. pylori survival. Mutation is acxABC led to reduce the colonization significantly in the stomach of mice [H. pylori to colonize in gastric mucosa.Acc_20 was found to contain SCOT complex associated with citric acid cycle and  of mice . So, AcchofC, hofD and lpxB were found to be present as an accessory island. LPS is present in the cellular membrane of all Gram-negative bacteria and plays a crucial role in pathogenesis. LPS has three distinct parts, a core composed of oligosaccharide, lipid A and O antigen. Lipid A is able to trigger fatal immune reaction even in very low concentration, thus known as endotoxin [x and Ley) are capable of interacting with DC-SIGN. It is a type II C-type lectin present on the dendritic cells of gastric epithelium. This interaction signal down-regulates the inflammatory cascade [H. pylori infection. Thus this GI is also associated with the disease prognosis indirectly. GC is a multifactorial disease which includes genetic and environmental factors. The high salt in-taking diet habit was found to be linked with increase the virulence of H. pylori. The virulence cagA positive strains were found to be enhanced with heavily salted diet in mice. It elevated the H. pylori colonization in the gastric mucosa and led to greater depletion of parietal cell in mice, thus facilitating the development of GC [cagA negative strains. So, rich salt diet promotes the GC prognosis in H. pylori infected people. Acc_14 was discovered to have a number of genes whose overexpression has been linked to a high-salt diet, for example, amiE, lysA. The exact function of these proteins in GC prognosis not yet understood.LPS biosynthesis genes such ndotoxin . The corndotoxin . The Lew cascade . This sunt of GC . This reH. pylori is critically influenced by flagellar motility. The fliD mutant strain was completely non-motile, was unable to colonize the stomach mucosa of host mice [fliD operon is linked to flagellar assembly and biofilm formation was found on Core_7. Many bacteria employ flagella to assist swarming, adhesion and biofilm formation. In biofilms, H. pylori may be able to withstand environmental challenges such stomach acidity and ROS produced by phagocytic cells. The bacteria can evade antibiotic therapy by living in a biofilms, leading to chronic infection [fliD) was found to be present across all the H. pylori strains and associated with essential function like flagellar biosynthesis and biofilm formation. It can be considered as a good target for development of a new therapeutic to combat with the H. pylori infection.The colonization potential of ost mice . The flinfection . FlagellC. jejuni, which is the most common cause of food poisoning in Europe and the United States [tilS, an essential protein for viability. TilS along with dus work in tRNA processing. Apart from this, tilS co-expresses with ddl which is involved in the peptidoglycan biosynthesis pathway. Thus, putative toxin–tilS interaction indirectly helps in tRNA processing, cell wall biosynthesis and vancomycin resistance. Generally, enterotoxins showed a marked effect in the gastrointestinal tract by increasing the chloride permeability of the intestinal mucosal cells [Characterization of accessory GI (Acc_31) led to the identification of a putative labile enterotoxin. Phylogenetic analysis suggested that this toxin was horizontally acquired from another helical-shaped, Gram-negative bacterium d States . Furtheral cells . The unial cells . The celH. pylori F30, H. pylori F16 and H. pylori F32) contain type II TAs which belong to Vap family and encoded by bicistronic operon. VapD was present in 60% of the H. pylori genomes [Design-Island-II identified these TAs in 29 strains. The lengths of the GI in 26 strains were <10 kb, due to deletion of neighboring gene content. Therefore, we studied this GI only in the aforementioned strains. Docking of vapD and its antitoxin forms a stable complex with minimum free energy. The antitoxin binds to vapD in the post-translational stage and inhibits its expression. The exact biological role of the vapD protein is yet to be established. Several reports suggest VapD in Haemophilus influenzae, acts as a toxin and in Rhodococcus equi helps in survival through acid tolerance [H. pylori in the gastric mucosa for a long time leads to the formation of severe lesions. Integrating all the reports we can presume, under stress and an unfavorable environment the partner antitoxin represses its own regulation, thus aiding the virulence of vapD.Toxin–antitoxin genes are often inherited through HGT . Three F genomes . Design-olerance ,65. In aolerance ,67. It hDesign-Island-II. This method is an unsupervised method, uses Monte-Carlo statistical test on randomly selected segments of a chromosome. It calculates the oligonucleotide frequencies and performs a statistical test to identify the GIs. Other sequence composition-based approaches, like PredictBias and MSGIP showed comparable prediction with Design-Island-II. In contrary, comparative genomics approach (IslandPick) and hybrid approach (IslandViewer 4) predicted GIs were somewhat different from Design-Island-II predicted results. However, experimental validation of these predicted GIs and their functional implication in disease pathogenesis need to be explorted to get a better insight on the role of these GIs.Various other GI prediction tools were used to validate the identified GIs by the  cagA and vacA virulent genes, it is equally important to study other virulence factors, as these have direct or indirect impact in the chronicity of gastric diseases like, some of them were linked to induce ulceration or bacterial colonization, while others aided in biofilm development, allowing bacteria to elude therapy and promote persistent infection.Finally, we can conclude, along with well-studiedClick here for additional data file.


Escherichia coli. Thus, the objective of this study was to characterize E. coli and detect its virulence factors and antibiotic resistance from raw milk samples.Raw milk is considered an essential source of nutrition during all stages of human life because it offers a valuable supply of protein and minerals. Importantly, milk is considered a good media for the growth and contamination of many pathogenic bacteria, especially food-borne pathogens such as E. coli using different biochemical tests, IMViC tests, serotyping to detect somatic antigen type, and molecularly by polymerase chain reaction (PCR) tests. The presence of different virulence and antimicrobial genes , and tetB genes) in E. coli isolates was evaluated using PCR.Raw milk samples (n=100) were collected from different localities in Qena, Egypt, and investigated for the presence of E. coli. Depending on serology, the isolates were classified as O114 (one isolate), O27 (two isolates), O111 (one isolate), O125 (two isolates), and untypeable (five isolates) E. coli. The sequencing of partially amplified 16S rRNA of the untypeable isolates resulted in one isolate, which was initially misidentified as untypeable E. coli but later proved as Enterobacter hormaechei. Moreover, antibacterial susceptibility analysis revealed that nearly all isolates were resistant to more than 3 families of antibiotics, particularly to b-lactams, clindamycin, and rifampin. PCR results demonstrated that all E. coli isolates showed an accurate amplicon for the blaTEM and tetA(A) genes, four isolates harbored eae gene, other four harbored tetB gene, and only one isolate exhibited a positive stx2 gene.The results demonstrated that 10 out of 100 milk samples were contaminated with E. coli as a harmful pathogen of raw milk using 16S rRNA sequencing, phylogenetic analysis, and detection of virulence factors and antibiotic-resistant genes.Our study explored vital methods for identifying  Escherichia coli is a facultative anaerobe and one of the normal inhabitants in the human and animal intestinal tracts [E. coli cause many diseases [E. coli strains have been categorized using different antibodies for perceiving surface antigens correlated to “183 O-groups (lipopolysaccharide) and 53 H-types (flagellar antigen)” [E. coli is differentiated into several pathotypes such as enterotoxigenic E. coli (ETEC) (O27), enterohemorrhagic E.coli (EHEC) (O111), and enteropathogenic E.coli (EPEC)” [l tracts . Howeverdiseases . Recentl (EPEC)” . In geneE. coli produce several effective toxins, such as Shiga-like toxins. These Shiga toxin-producing E. coli (STEC) contribute toward gastroenteritis, bloody diarrhea, and uremic syndrome in infected humans [E. coli combined with plasmid-encoded enterohemolysin (hlyA), which is frequently associated with acute sickness in individuals [eaeA), which is responsible for attachment, adherence, and clustering epithelial cell surfaces [E. coli possess these virulence genes. However, the procurement of one or more virulence genes does not place bacteria in a harmful grade if that strain has not harbored the proper virulence gene that initiates disease in specific species [E. coli isolates from human, animal, and environmental sources.” Antimicrobial resistance has been observed as a severe problem worldwide [E. coli strains diverge in different environments [E. coli, specifically those from animal sources [Dissimilar strains of pathogenic d humans . Other vividuals , and alssurfaces . Polymer species . “The diorldwide . Howeverronments . Many au sources .E. coli while causing several diseases and to optimize uniplex PCR assays for their detection in milk samples.Therefore, the main objectives of this study are to apply a broader array of virulence and antimicrobial-resistant genes that are known to arise in different pathotypes of The study was approved by the Animal Ethics Committee for Veterinary Research, Faculty of Veterinary Medicine, South Valley University, Qena, Egypt.The study was conducted from February to August 2019. A total of 100 milk samples were collected from different sources  in Qena, Egypt. These samples were collected under aseptic conditions in a clean, sterile 15 mL falcon tube and transferred instantly in an icebox to the bacteriological laboratory in the Department of Microbiology, Faculty of Veterinary Medicine, South Valley University. One milliliter of milk samples were fed in 9 mL buffer peptone water (Oxoid) and incubated at 37°C for 18-24 h. Subsequently, a sterile loop was used to transfer bacteria from the inoculated buffer peptone water and was inoculated on a MacConkey agar plate (Oxoid). Plates were incubated at 37°C for 24 h. The suspected colonies were inoculated in eosin methylene blue (Oxoid). After the procedure, green metallic sheen colonies were selected for biochemical identification using the IMViC reaction and triple sugar iron test .E. coli isolates were serogrouped according to a previous study [E. coli isolates was performed by commercially available kits , which combined 8 polyvalent sera and 43 monovalent sera.us study . The serAntimicrobial susceptibility tests were conducted using the disk diffusion method following the guidelines of a previous research . A rulerDNA extraction was performed using the QIAamp DNA Mini kit  with changes to the manufacturer’s recommendations. Briefly, 10 μL of proteinase K and 200 μL of lysis buffer were added to 200 μL of the bacterial suspension, and this mixture was then incubated at 56°C for 10 min. After incubation, the lysate was mixed with 200 μL of 100% ethanol. The mixture was then rinsed and centrifuged after the manufacturer’s recommendations. Nucleic acid was washed with 100 μL of elution buffer accompanied within the kit.The extracted bacterial DNA and controls were amplified with 12.5 μL of Emerald Amp Max PCR Master Mix , 1 μL of each primer (20 pmol concentration), 6 μL of DNA template, and 4.5 μL of water to reach a final volume of 25 μL. DNA was amplified  in a the16S rRNA of untypeable E. coli strains were sequenced in the forward and reverse directions, as well as in separate reactions, using primers of 16S rRNA, according to the manufacturer’s protocol using the following kits: Big Dye Terminator V.3.1 Cycle sequencing kit (Applied Biosystems), the sequencing cycle purification kit, DyeEx 2.0 Spin kit (Qiagen), and Hi-Di-ionized formamide (Applied Biosystems). DNA sequences were obtained from the Applied Biosystems 3130 . All the obtained 16S rRNA gene sequences were submitted to the GenBank. The multiple alignment algorithms in MegAlign  were used to perform the sequence alignment of isolates.Purified PCR products of partially amplified 16S rRNA gene nucleotide sequence, was a tribute for the untypeable isolates to demonstrate these isolates’ identities and reference strains logged in GenBank using MegAlign from the Lasergene package version 7 (Dnastar).A phylogenetic tree, which relied on the hly, eaeA, blaTEM, tetA(A), and tetB were used in a 25 μL reaction containing 12.5 μL of Emerald Amp Max PCR Master Mix (Japan), 1 μL of each primer, 4.5 μL of water, and 6 μL of DNA template. The reaction was conducted in an Applied Biosystem 2720 thermal cycler , 1 μL of each primer (concentration of 20 pmol), 13 μL of water, and 8 μL of DNA template. The reaction was conducted in a thermal cycler (Applied Biosystem 2720) [Primers for em 2720) -20  in 1× TBE buffer at room temperature (30ºC) using a gradient of 5 V/cm. For gel analysis, 20 μL of each product was loaded in each gel well. A 100 bp DNA ladder (Qiagen) was used to find out the amplicon sizes. The gel was visualized by a gel documentation system .E. coli isolates from the raw milk samples, using antisera against the O-antigen, exhibited that one isolate was O111, one isolate was O27, one isolate was O114, two isolates were O125, and the other five isolates were untypeable (We isolated from 10 out of 100 raw milk samples (10%). Four samples were brought from markets, three samples were brought from dairy farms, and three samples were obtained from farmers’ houses. The serology results of the 10 typeable .E. coli isolates was determined against 12 different antibiotics according to Clinical Laboratory Standards Institute [E. coli isolates did not show any sensitivity for oxacillin, penicillin, rifampin, and clindamycin. A total of nine isolates were resistant to erythromycin, and six isolates were resistant to trimethoprim. Moreover, five isolates were resistant to trimethoprim-sulfamethoxazole, chloramphenicol, nitrofurantoin, and gentamicin; furthermore, seven isolates did not have any zone of inhibition against tetracycline and four isolates were resistant to nalidixic acid. Four isolates were sensitive to both trimethoprim and trimethoprim-sulfamethoxazole. Three isolates were sensitive to chloramphenicol and nitrofurantoin.The antibiotic susceptibility profile of the 10 nstitute . All 10 Two isolates were resistant to tetracycline and gentamicin, whereas only one isolate was sensitive to erythromycin. Six isolates cleared intermediary susceptibility toward nalidixic acid, three isolates exhibited intermediate susceptibility to gentamicin, two isolates showed intermediary susceptibility toward chloramphenicol and nitrofurantoin, and only one isolate exhibited intermediary susceptibility toward trimethoprim-sulfamethoxazole and tetracycline .E. coli DNA in 10 isolates using housekeeping gene primers (16S rRNA). The presence of different virulence genes  and antibiotic-resistant genes , and tetB) was then evaluated in the 10 isolates (PCR results confirmed the presence of isolates .E. coli isolates (O125) and two untypeable E. coli isolates were positive for the eaeA gene (E. coli isolate (O125) was positive for the hly gene  with similarity percentages of 98.9%, 98.1%, and 98.3%, respectively. The last isolate identified phenotypically as E. coli (isolate 5) exhibited the most similarity  with E. hormaechei (MF768986)  .E. coli. These results are nearly similar to those of Kumar and Prasad [E. coli. da Silva et al. [E. coli from 22.1% of milk samples in Brazil. Disassa et al. [E. coli from 33.9% of cow milk samples in Ethiopia. These studies share the same concern as our research that E. coli contamination of raw milk samples is a growing challenge.The phenotypic identification of raw milk samples revealed a concern with maintaining proper hygienic conditions at the sampling sites. Among the 100 milk samples collected, 10 (10%) were contaminated with d Prasad , who foua et al.  isolateda et al.  isolatedE. coli features many lipopolysaccharides distinguished by the O-antigen. The enucleating of the O-antigen from the wall of bacterial cell attenuate bacterial pathogenicity in suggesting the O-antigen, which plays a key role in host-pathogen interactions [E. coli since the 1940s [E. coli isolates sampled from raw milk sources in our study, five isolates were serotyped as O111, O27, O114, and O125, and five strains were untypeable by agglutination. The identified isolates were differentiated according to a previous study [E. coli (ETEC) (O27), enterohemorrhagic E. coli (O111), and enteropathogenic E. coli (EPEC) (O114 and O125). Our results are related to the results obtained by the previous studies [E. coli serogroups in cattle [E. coli isolates were untypeable in the agglutination test. This highlights one of the challenges of serological methods, as discussed by Delannoy et al. [The outer layer of ractions . Corresphe 1940s . Based ous study  as enter studies . Severaln cattle ,29,30. Pn cattle -33. As sy et al.  who founy et al. .E. coli [E. coli strain but was later confirmed as an isolate of E. hormaechei (accession no. MN066412). Garbaj et al. [E. coli strains identified using conventional methods, only 11 strains were confirmed to be E. coli using 16S rRNA sequencing. This result suggests the importance of using molecular methods to validate the isolate information obtained from conventional serological techniques.The DNA sequencing of 16S rRNA can aid in the identification of E. coli , especiaj et al.  found thE. coli strains earned from milk samples by the neighbor-joining tree found that there was a high degree of similarity between our isolates (similarity of 95.5-100%), as all of these species originated from the Enterobacteriaceae family [E. coli (MN064835) was related to two reference E. coli strains (MN006357 and KY711198). Both strains are uropathogenic E. coli (UPEC) strains [E. coli isolates MN066319, MN064861, and MN06483 were found to be related to a reference strain (E. coli AP019675), which is an extended-spectrum b-lactamase E. coli (ESBL) strain [E. coli isolates in raw milk are unsurprising. Nevertheless, this observation highlights the importance of performing a microbiological analysis of milk to verify that there is no public health risk to consume the milk. This is particularly important to curb the outbreaks of food-borne illnesses, which were formerly linked to the consumption of milk and dairy products that had been contaminated with pathogenic bacteria or their toxins [The additional appraisal of the 16S rRNA sequences of e family . The seq strains . Moreove) strain . Most of) strain ,40. Thusr toxins .stx2 gene was found in only one isolate (untypeable E. coli), whereas the stx1 gene was not harbored by any of the 10 isolates. Our result matches the previous studies [stx1 and stx2 was noted by Zschock et al. [E. coli obtained from cows. This may be attributed to the repeated turnover of serotypes of E. coli, with many being isolated only sporadically from a herd [et al. [stx genes detected, according to the distribution of STEC.STEC (Stx) strains were identified from cattle farms more than 3 decades ago. Studies implemented in several regions have shown that 10–80% of cattle may harbor STEC ,42,43. I studies ,44 but d studies ,46. Almok et al.  in the fm a herd . Further [et al.  confirmeE. coli utilizes various virulence factors to colonize and infect host cells. One factor is intimin (encoded by the eaeA gene), which plays an important role in the pathogenesis of attaching-and-effacing lesion, which facilitates the colonization of host tissues [eaeA gene was detected in four E. coli isolates (40%). Two isolates were EPEC of serotype O125 and O114, and the other two isolates were untypeable E. coli. Many authors have previously revealed that EPEC strains harbor the eaeA gene [eaeA gene was detected in 4% of E. coli isolates in a previous study [et al. [As it pertains to pathogenesis,  tissues . In our aeA gene ,52. The us study  and 25%  [et al. .E. coli to promote pathogenesis is the liberation of hemolysin, a pore-forming cytolysin (encoded by the hlyCABD gene). In our study, we illustrated that only one isolate belonging to EPEC harbored the hly gene. EPEC strains, UPEC strains, ETEC strains, and STEC strains carried the hly gene, which is encoded on a plasmid reported by Burgos [A second key virulence factor used by y Burgos .E. coli isolates obtained from the raw milk samples. The propagation of antibacterial resistance is the leading cause of many health problems worldwide. In this study, we found that a high percentage (100%) of E. coli isolates were resistant to penicillin, oxytetracycline, doxycycline, rifampicin, trimethoprim/sulfamethoxazole, and tetracycline. Multidrug resistance (resistance to more than 3 classes of antibiotics) was also observed in several of our E. coli isolates. The isolation of multidrug-resistant E. coli isolates is not surprising as this phenotype has been reported for E. coli in other studies [E. coli isolates obtained from milk samples were resistant to antibiotics, mainly to b-lactam antibiotics [After evaluating the existence of genes encoding key virulence factors, we next assessed the antibiotic resistance profile of the 10  studies ,56. In aibiotics ,58.tetA(A) and tetB) were the most prevalent. This finding is concerning because tetracycline is one of the most important antibiotics used in Egypt [Bacterial resistance to antibiotics may occur by an impulsive mutation in the target gene(s) due to using antibiotics in therapy or enhancing growth in animals. These antibiotic-resistant bacteria can be spread in the environment when infected animals defecate, thus contaminating soil and agricultural products. If the raw products are consumed, then the resistant bacteria may consequently colonize and infect humans . Among tin Egypt ,61.E. coli. Focusing on ESBLs, in this study, the blaTEM gene was detected in all E. coli isolates. The prevalence of blaTEM found in our E. coli isolates matches results obtained by the previous studies [E. coli isolate harbored blaTEM.In addition to resistance to tetracycline, the presence of ESBL’s confers resistance to many antibiotics, such as aztreonam, related oxyimino-b-lactams, cephalosporins, and penicillin. There is a wide variety of ESBLs, including plasmid-mediated resistant to ampicillin, oxacillin, and cefotaxime . The mec studies ,64, who E. coli can arise through gene transfer in the environment or the human and animal guts. The genes can be found either on the same plasmid or separately on bacterial chromosomal DNA and plasmids. These genes might be coselected in response to antibiotic pressure, hence aggravating the hazard imposed by E. coli on food safety [Our study demonstrated a relationship between the resistance to drugs and the presence of virulence factors. The attainment of antibiotic resistance and virulence factors in d safety ,66.E. coli is considered one of the most common food-borne pathogens that can be transmitted to humans by drinking raw milk. Some strains of E. coli harbor some virulent genes eae, hyl stx2 genes, antibiotic-resistant genes blaTEM, tetA(A), and tetB. Furthermore, they showed marked resistance to several antibiotics commonly used in animals and humans in Egypt (MDR). Therefore, much more attention should be paid to hygienic measures during the milking process in dairy farms and the prudent use of antibiotics supported by antibiogram tests before drug administration in dairy farms.According to this study’s findings, WY and HMAM: Designed the study, drafted and critically revised the manuscript, collected samples, and did laboratory works. SH: Revised the manuscript and interpretation of the data. All authors read and approved the final manuscript.


Angiosarcoma of the Posterior Mediastinum is a rare entity. We herein report a case of a giant posterior mediastinal Angiosarcoma. A 54-year female presented with a one-year history of epigastric pain. Her upper gastrointestinal tract endoscopy revealed no abnormality. Chest x-Ray showed a shadow in the hilar area. A computed tomographic scan of the Thorax (CT) and MRI Scan showed a mass in the Posterior Mediastinum. CT-guided biopsy revealed the tumor of vascular origin. The tumor was completely resected. Post-operative recovery was uneventful. After 14 months follow up patient is disease-free, and a CT scan of the chest showed no recurrence. •A 54 years old female nonsmoker presented with a one-year history of epigastric pain.•C-XRAY showed a large right hilar mass.•CT Scan of thorax showed a large heterogeneous mass measuring 13.5 × 8.0cm.•CT guided biopsy showed tumor of vascular origin.•Radical surgical resection was performed with excellent outcome. Most commonly, it occur as sporadic cutaneous lesions of the scalp, superficial connective tissues. Rarely do they involve deep soft tissues and body cavities. Weiss et al. reported after a review of 300 cases of Angiosarcoma that one-third of them occurs in the cutaneous layers, most commonly in the head and neck, one-fourth in the soft tissues, and rest in the liver, spleen, breast, bone, and heart ,2. Angio2HCG), and Lactate dehydrogenase (LDH) were within normal limits. Her chest X-ray showed an increased right hilar shadow A 54 years old female nonsmoker presented with a one-year history of epigastric pain. There was no history of cough, shortness of breath, loss of weight, or appetite. On clinical examination, there was no jaundice, edema, or lymphadenopathy. She has no specific past medical history. On auscultation, there was decreased air entry in the right hemithorax. Her ultrasound of the abdomen and upper gastrointestinal endoscopy were reported normal by the referral hospital. Her basic blood investigations, white cell count, liver and renal panels were within normal range. Tumor markers including alpha-fetoprotein,beta-human gonadotropin , operating surgeon and drafted the article.Othman M Al Fraih, wrote abstract and case details.Meenal A Al Abdulhai, structured abstract.Ahmed Alshaer, highlights.Burair Al Jassas, searched references.Hisham Mamon images and legends.Yousif AL Qahtani, wrote background.Miral Mashour Histopathologist.Abdullah M Al Ghamdi wrote part of discussion.1.Name of the registry: Research registry.2.Unique Identifying number or registration 7368.3.http://www.researchregistry.com/browse-the-registry#home/.Hyperlink to your specific registration (must be publicly accessible and will be checked): Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request”.Ikram ul haq Chaudhry.Not commissioned, externally peer reviewed.No conflict of interest and there was no funding or financial assistance in this case.


Public health and social measures have been implemented around the world in a bid to prevent the spread of COVID-19. Public compliance with these measures is key in successfully controlling the pandemic. This online survey assessed the compliance and attitude of adults residing in the southern African Kingdom of Eswatini to government protection, activity and travel measures aimed at controlling the spread of COVID-19. A rapid online survey, comprising of 28 questions, was administered in May 2020. More than 90% of respondents knew the virus could kill anyone and most respondents (70%) reported to be compliant to public health and social measures. Females, those who did not use public transport and those aged 30 years and above were significantly (p<0.01) more compliant, particularly to protective and travel measures. Social media, television and official government websites were the primary source of ongoing COVID-19 information for respondents of this online survey, and these methods should continue to be employed to reach the public who regularly use the internet. More than half of essential workers who responded to the online survey reported to have their own personal protective equipment; however, 32% either did not have any protective equipment or shared their equipment with other staff members. Due to the survey being online, these results should not be generalised to populations of low socioeconomic status. COVID-19 disease, caused by the SARS-COV-2 coronavirus, was identified in 2019 . COVID-1rd June 2021, 19 COVID vaccines are in the World Health Organisation Emergency Use List evaluation process . Ap. Ap68]. The Government of Eswatini introduced public health and social measures to control the spread of COVID-19 disease , 45, 69.This study found a variety of variables that significantly influenced compliance including gender, age using public transport, and beliefs surrounding COVID-19. Gender differences in compliance has been found in other studies, where males were significantly associated with risky behaviours related to COVID-19  . This stAge differences in compliance with COVID-19 prevention measures has been shown in the U.S. , EcuadorPublic transport, especially when crowded, can be associated with the spread and acquisition of respiratory infectious diseases , 30. At Among the many public health and social measures implemented to stop the spread of SARS-COV-2 by governments across the world was for people to stay at home and work from home where possible, with the exception of essential workers who were needed to provide medical care, food and other essential services to the population . In EswaStudies have found the number of people in a household to be positively associated with acquiring COVID-19  and MERSWhile, other studies showed that beliefs and knowledge of COVID-19 were correlated with compliance to preventive measures , 16, 21,st June 2020, the survey was closed. Therefore, the online survey was live on the SurveyMonkey website for a limited time thereby restricting the number of participants. Our study uses data from respondents self-reporting using an online survey and therefore can be affected by social desirability bias. Whilst this was not controlled for in the study questions, the survey was anonymous, voluntary and respondents could backtrack and change their answers before submitting their survey, which has been associated with a decrease in social desirability bias [This study has limitations that should be considered when interpreting the data. Due to travel restrictions, traditional door-to-door or face-to-face surveys were not employed. The high costs of communication and the lack of available funding for this study made SMS or telephonic surveys prohibitively expensive, and sampling would be restricted to the limited database(s) we could employ. Therefore, this study used an online survey coupled with website, traditional media and social media outreach to recruit participants resident in the four regions of Eswatini. This study targeted adults who use the internet ; the survey was not designed to capture the perceptions, attitudes and behaviour of those who did not use such technology during the time the survey was live and cannot be considered as representative of the general public. As described above, approximately half of the population in Eswatini is thought to have access to the internet and a quarter of the population are active monthly social media users . Althougity bias .Our online survey indicates that between 14–31 May 2020, adult residents in the southern African Kingdom of Eswatini with access to the internet were mostly compliant to government public health and social measures. Females, public transport users, older age groups and those with medical aid reported greater compliance to these COVID-19 prevention measures. Essential workers were overall compliant to government measures; however, the provision of dedicated personal protective equipment was highlighted as a challenge for them. Most respondents to our survey had the capacity to isolate household members, which is important as changes to the Government’s testing and treating strategy implemented two months after the close of survey saw persons presumed to have COVID-19 to be isolated at home, and only those with moderate to severe symptoms to be hospitalised.S1 Text(PDF)Click here for additional data file.S1B-E FigB. Compliance of personal protective measures by gender. C. Travel compliance to village homestead by gender. D. Travel frequency compliance by gender E. Travel compliance during public holidays by gender. WHS = washing hands and sanitising. SD = social distancing. SH = staying home. WM = wearing a mask. NOA = none of the above. NSH = no, I stayed home. NVH = no village homestead. LVH = lives at village homestead. Y12 = yes, once or twice. Y3+ = yes 3 or more times. DNA = did not answer.(TIF)Click here for additional data file.S2C-G FigC. Compliance of personal protective measures by essential workers. D. Travel compliance to village homestead by essential workers. E. Travel frequency compliance by essential workers F. Travel compliance during public holidays by essential workers. G. Overall compliance score for essential workers. Abbreviations: FA = food and agriculture, RCGS = retail and consumer goods supplier, NI = network infrastructure, MH = medical and health, FSM = forestry and saw mills, ITST = IT systems and telecommunications, FI = finance and insurance, TH = tourism and hospitality, Comm = communications , HS = hardware stores, NPO = non-profit organisations, Edu = education, O = other, WHS = washing hands and sanitising. SD = social distancing. SH = staying home. WM = wearing a mask. NOA = none of the above. NSH = no, I stayed home. NVH = no village homestead. LVH = lives at village homestead. Y12 = yes, once or twice. Y3+ = yes 3 or more times. DNA = did not answer, Yes = essential worker (n = 129), No = not essential worker (n = 328).(TIF)Click here for additional data file.S3C-F FigC. Compliance of personal protective measures by public transport users. D. Travel compliance to village homestead by public transport users. E. Travel frequency compliance by public transport users F. Travel compliance during public holidays by public transport users. Abbreviations: WHS = washing hands and sanitising. SD = social distancing. SH = staying home. WM = wearing a mask. NOA = none of the above. NSH = no, I stayed home. NVH = no village homestead. LVH = lives at village homestead. Y12 = yes, once or twice. Y3+ = yes 3 or more times. DNA = did not answer. Yes = public transport user (n = 102), No = not public transport user (n = 302). Fifty-three respondents declined to answer if they did or did not use public transport.(TIF)Click here for additional data file.S4B-E FigB. Compliance of personal protective measures by age C. Travel compliance to village homestead by public transport users. D. Travel frequency compliance by public transport users E. Travel compliance during public holidays by public transport users. Abbreviations: WHS = washing hands and sanitising. SD = social distancing. SH = staying home. WM = wearing a mask. NOA = none of the above. NSH = no, I stayed home. NVH = no village homestead. LVH = lives at village homestead. Y12 = yes, once or twice. Y3+ = yes 3 or more times. DNA = did not answer. One respondent declined to identify their age.(TIF)Click here for additional data file.S5C-D FigD. Sources of media used to continue staying up to date about COVID-19, by age. SM = social media, N = newspaper, TV = television, R = radio, FFCN = family friends colleagues or neighbours, MGW = Ministry of health or Government of Kingdom of Eswatini website, O = other, I = internet.(TIF)Click here for additional data file.S6 FigLight grey = observed values. Imputed values = dark grey.(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Apis cerana–Nosema ceranae interaction is very limited, though A. cerana is the original host of N. ceranae. Apis cerana cerana is a subspecies of A. cerana and a major bee species used in the beekeeping industry in China and other countries. Here, the effective infection of A. c. cerana workers by N. ceranae was verified, followed by transcriptomic investigation of host responses. Furthermore, immune responses between A. c. cerana and Apis mellifera ligustica were deeply compared and discussed. In total, 1127 and 957 N. ceranae-responsive genes were identified in the infected midguts at 7 d post-inoculation (dpi) and 10 dpi, respectively. Additionally, DEGs in workers’ midguts at both 7 dpi and 10 dpi were associated with six cellular immune pathways and three humoral immune pathways. Noticeably, one up-regulated gene was enriched in the NF-κB signaling pathway in the midgut at 10 dpi. Further analysis indicated that different cellular and humoral immune responses were employed by A. c. cerana and A. m. ligustica workers to combat N. ceranae. Our findings provide a foundation for clarifying the mechanisms regulating the immune response of A. c. cerana workers to N. ceranae invasion and developing new approaches to control bee microsporidiosis.Currently, knowledge regarding Apis cerana cerana workers at 7 d post-inoculation (dpi) and 10 dpi with Nosema ceranae and corresponding un-inoculated midguts. PCR identification and microscopic observation of paraffin sections confirmed the effective infection of A. c. cerana worker by N. ceranae. In total, 1127 and 957 N. ceranae-responsive genes were identified in the infected midguts at 7 dpi and 10 dpi, respectively. RT-qPCR results validated the reliability of our transcriptome data. GO categorization indicated the differentially expressed genes (DEGs) were respectively engaged in 34 and 33 functional terms associated with biological processes, cellular components, and molecular functions. Additionally, KEGG pathway enrichment analysis showed that DEGs at 7 dpi and 10 dpi could be enriched in 231 and 226 pathways, respectively. Moreover, DEGs in workers’ midguts at both 7 dpi and 10 dpi were involved in six cellular immune pathways such as autophagy and phagosome and three humoral immune pathways such as the Toll/Imd signaling pathway and Jak-STAT signaling pathway. In addition, one up-regulated gene (XM_017055397.1) was enriched in the NF-κB signaling pathway in the workers’ midgut at 10 dpi. Further investigation suggested the majority of these DEGs were engaged in only one immune pathway, while a small number of DEGs were simultaneously involved in two immune pathways. These results together demonstrated that the overall gene expression profile in host midgut was altered by N. ceranae infection and some of the host immune pathways were induced to activation during fungal infection, whereas some others were suppressed via host–pathogen interaction. Our findings offer a basis for clarification of the mechanism underlying the immune response of A. c. cerana workers to N. ceranae infection, but also provide novel insights into eastern honeybee-microsporodian interaction.Here, a comparative transcriptome investigation was conducted based on high-quality deep sequencing data from the midguts of  Honeybees are a pivotal pollinator for a great number of wild plants and crops throughout the world . In addiNosema ceranae is a spore-forming unicellular and obligate intracellular fungal parasite that exclusively infects the midgut epithelium cells and not all epithelial cells of the adult honeybee, leading to bee microsporidiosis  was simultaneously enriched in autophagy and lysosome  = −11.16) and lysosomal aspartic protease  = −1.17) were simultaneously enriched in autophagy and lysosome  enriched in this cellular immune pathway were down-regulated  = −10.81) and 10 dpi (log2(fold change) = −12.19). These results suggested that the aforementioned two cellular immune pathways were suppressed by N. ceranae at different stages of fungal infection.In this current work, Venn analysis displayed that the numbers of specific up-regulated genes were 297 and 245, which were much less than those (706 and 582) of specific down-regulated genes in AcCK1 vs. AcT1 and AcCK2 vs. AcT2; additionally, only 124 DEGs were shared by the above-mentioned two comparison groups, including 30 up-regulated and 94 down-regulated genes. It is speculated that some DEGs in d 10 dpi . In addiA. m. ligustica workers at 7 dpi and 10 dpi with N. ceranae, respectively, suggesting that that apoptosis pathway was activated to combat N. ceranae infection [N. ceranae was shown to suppress apoptosis in host midgut epithelial cells [N. ceranae-tolerant A. mellifera workers could escape parasitic manipulation of apoptosis [A. c. cerana worker midguts responding to N. ceranae invasion.One up-regulated gene was previously discovered to be enriched in apoptosis in the midguts of nfection . Intrigunfection . This stnfection ,80. In wal cells ,19,28. Hpoptosis . That fiDrosophila and mosquito. The NF-κB pathway, a conserved signal transduction pathway present in nearly all mammalian cell types, is involved in various biological processes including innate and adaptive immunity, survival, development, proliferation, and cell apoptosis [κB pathway in N. ceranae-challenged A. c. cerana workers’ midgut at 10 dpi, and one up-regulated gene and one down-regulated gene were enriched in the Toll-like receptor signaling pathway  that are uniquely active against different pathogens [A. m. ligustica workers at both 7 dpi and 10 dpi with N. ceranae, one up-regulated gene was enriched in the NF-κB pathway, implying that this humoral immune pathway was activated by N. ceranae infection [κB pathway may play a pivotal role in immune responses of both eastern honeybee workers and western honeybee workers to N. ceranae invasion. In addition, five genes enriched in the Jak-STAT pathway were up-regulated in workers’ midgut at 7 dpi, while three DEGs in workers’ midgut at 10 dpi were engaged in this humoral immune pathway, including two up-regulated genes (Drosophila [Anopheles gambiae responding to bacterial infection [A. c. cerana workers’ midguts was activated due to N. ceranae infection. Intriguingly, there was no DEG enriched in the Jak-STAT pathway in A. m. ligustica workers’ midguts in response to N. ceranae infection [A. c. cerana workers and A. m. ligustica workers to battle the same fungal parasite, N. ceranae.After fungal infection, insect humoral immunity is induced to synthesize AMPs and activate the prophenoloxodase system, which can work together to inactivate or kill the invaders . Currentpoptosis ,83. Here pathway B, wherea pathway B. Drosopathogens ,85,86,87athogens ,89. In tnfection . These red genes B. The Jaosophila ,91. Jak-osophila . This imnfection . It has nfection . TogetheA. c. cerana worker response to N. ceranae invasion. That effort can be expected to provide knowledge for the development of effective and environmentally friendly strategies to control bee nosemosis.Additional work is needed to decipher the function of key immune genes with differential expression in A. c. ceranae workers’ midguts to N. ceranae invasion were comprehensively investigated at a transcriptome-wide level. These results demonstrated that the overall gene expression profile in host midgut was altered by N. ceranae invasion and that some host immune pathways such as the autophagy and Jak-STAT pathway were induced to activation during fungal infection, whereas some were suppressed via host–pathogen interaction. Our findings not only offer a basis for clarification of the mechanism underlying the immune response of A. c. cerana to N. ceranae infection, but also provide novel insights into eastern honeybee–microsporidian interaction.In the present study, cellular and humoral immune responses of