Salmonella (NTS) is one of the important bacteria that cause foodborne diseases and invasive infections in children and elderly people. Since NTS infection is difficult to control due to the emergence of antibiotic-resistant species and its adverse effect on immune response, the development of a vaccine against NTS would be necessary. This study aimed to develop a multi-epitope vaccine against the most prevalent serovars of NTS  using an immunoinformatics approach and targeting OmpA, OmpD, and enterotoxin (Stn).Non-typhoidal E. coli plasmid vector pET-30a (+) within restriction sites of HindIII and BamHI.Initially, the B cell and T cell epitopes were predicted. Then, epitopes and suitable adjuvant were assembled by molecular linkers to construct a multi-epitope vaccine. The computational tools predicted the tertiary structure, refined the tertiary structure and validated the final vaccine construct. The effectiveness of the vaccine was evaluated via molecular docking, molecular dynamics simulation, and in silico immune simulation. The vaccine model had good binding affinity and stability with MHC-I, MHC-II, and toll-like receptors  as well as activation of T cells, IgM, IgG, IFN-This study, for the first time, introduced a multi-epitope vaccine based on OmpA, OmpD and enterotoxin (Stn) of NTS that could stimulate T and B cell immune responses and produced in the prokaryotic system. This vaccine was validated in-silico phase which is an essential study to reduce challenges before in vitro and in vivo studies. According to the CDC report, about 40,000 cases are registered annually in the United States . Two major Salmonella serovar related to the foodborne disease are Salmonella enterica, serovar enteriditis and Salmonella enterica, serovar typhimurium which are known as NTS [Salmonella's enterotoxin (Stn) is responsible for diarrhea [Salmonella infection is difficult due to the bacteria’s tolerance to environmental stress, its multi-drug resistance and wide distribution [Salmonella invades macrophages and escapes from phagocytosis. Salmonella reduces germinal center formation and survival of IgG secreting-plasma cells. Since NTS infection remains a severe health problem, vaccine development is essential to avoid loss of immune responses [Non-typhoidal Salmonella (NTS) is a major cause of bacterial enteritis with 93.8 million diseases and 155,000 deaths each year [n as NTS . Salmonediarrhea , 7. Moreribution . AntibioS. Typhimurium and S. Enteritidis . Furthermore, researchers discovered that the outer membrane proteins (Omp) include a class of proteins known as porins, which induce antibody response and cell-mediated immunity [Recently, several vaccines candidate has been developed for NTS including live-attenuated, subunit-based, and recombinant antigen-based vaccines . The WT0immunity . The majS. Typhimurium and S. Enteritidis [OmpD and OmpA. Evidence indicated that OmpD and OmpA are the highly conserved and abundant porins in eritidis –15. SincS. Typhimurium and S. Enteritidis, as well as their enterotoxin (Stn), were investigated in order to construct bivalent multi-epitope vaccine.OmpD), but no commercial vaccine is available so far , 9, 16. S. Typhimurium and S. Enteritidis. Results of this analysis show more than 99% similarity in OmpA, OmpD of S. Typhimurium and S. Enteritidis while no considerable similarity was seen for Stn between these bacteria.First, the OmpA, OmpD and Stn were analyzed to identify their amino acid sequence similarity between According to IEDB server results, in total 4177 epitopes for CTLs and14413 epitopes for HTL were predicted. The best epitopes were chosen for further analysis based on their high binding affinity, antigenicity score, non-allergenicity, non-toxicity and immunogenicity as illustrated in Tables The linear B cell epitopes were predicted using ABCpred server with (16 mer) length and cut-of binding score > 0.51. With these criteria, epitopes with higher scores from OmpA, OmpD and Stn were chosen for vaccine construction, which is listed in Table Global population coverage analysis for 8 selected epitopes (4 CTL and 4 HTL) shows 100% and 95.85% of people respond to CTL and HTL epitopes respectively.Four epitopes were nominated for each HTL, CTL and B cell to construct vaccine. An adjuvant Griselimycin (APD ID: AP02688) was added to both N and C terminals of the vaccine sequence to increase immunogenicity. In addition, three linkers were used to link epitopes: GPGPG and AAY linkers were used to link B-cell, HTL and CTL epitopes respectively, and EAAAK linkers attach adjuvant to the N and C terminal of epitopes Fig. The result of vaccine's antigenicity was predicted to be 0.81, which defines its highly antigenic properties. In addition, the vaccine model on both AllerTOP and ToxinPred servers was non-allergen and non-toxic respectively.E. coli in vivo respectively. Additionally, the instability index value was 27.57 which indicates the vaccine is stable (> 40: instability). The aliphatic index was 87.09 which shows thermostability [The vaccine model was characterized by a molecular weight of 24.07 kDa with a theoretical isoelectric point (pI) score of 9.49. The vaccine model's half-life was estimated at 100 h for mammalian reticulocytes in vitro and more than 20 h and 10 h in yeast and tability  and GranA 3D vaccine structure was built by the I-TASSER server. According to 10 threading templates, 5 tertiary 3D structures were predicted with Z-scores (0.67–1.01) and confidence score (C-score: − 5 to − 4). The C-score is typically in the range of  − 5, 2), where high score indicates high confidence in the predicted model. Based on these criteria, the best model (C-score: -4) was selected for further analysis. Additionally, this model had TM score and root-mean-square deviation (RMSD) score of 0.19 ± 0.05 and 19.2 ± 2.0 Å respectively , RMSD (0.461), and MolProbity (2.117), clash value (11.1), Poor rotamers (0.6) and Rama value (89.8). This model was chosen for further investigation  to investigate the stability of the vaccine and TLR-1, TLR-2 and TLR-4 complex. The results are shown in Fig. The results of the immune system simulations produced by C-ImmSim server indicated a notable increase in immune responses that were comparable to the real immunological responses. The primary response was determined by an increase in IgM levels. In secondary and tertiary injections, it was seen that B-cell population, IgG1 + IgG2 antibodies, and IgG + IgM antibodies increased, while antigen levels decreased. In addition, the vaccine model could increase the TH population with memory cells and TC cells. IFN-γ and IL-2 production was also elevated after repeated exposure. These results validate the antigenic and immunogenic characteristics of the vaccine model Fig. .Fig. 6InE. coli K12 [The vaccine amino acid sequence after JCAT optimization had a length of 711 nucleotides. The CAI score was predicted 1.0 and the GC content was 56.6%, which indicates high expression in the coli K12 . FinallySalmonella is the most common cause of gastrointestinal disease in healthy people and invasive infections in patients with immune-compromised systems. From the past till now, vaccine development has been considered the most efficient and cost-effective strategy to prevent infectious diseases. For years, scientists have been trying to develop a NTS vaccine, however, there is no licensed vaccine currently available [vailable . Since wvailable . Therefovailable –24.S. Typhimurium based on an in-silico analysis. The authors selected B and T-cell epitopes found in TolA (membrane-spanning protein) as the most antigenic protein in S. Typhimurium. In another study, Priyadarsini et al. [Salmonella Omps are accessible for immune system, essential to cell communication, stable in high temperature and highly immunogenic, they have been considered as potential vaccine candidates in recent years. However, for NTS, no more investigation has yet been conducted. On the other hand, the pathogenicity of S. Typhimurium and S. Enteritidis were associated to enterotoxin (Stn) [salmonella enterotoxin. Accordingly, three major proteins OmpA, OmpD and Stn from two serovars S. Typhimurium and S. Enteritidis were selected for investigation. The NCBI database was used to retrieve protein sequences and BLASTp analysis reveals that OmpA, OmpD share more than 99% identity in S. Typhimurium and S. Enteritidis. As a result, to construct a multi-epitope vaccine, HTL, CTL and B cell epitopes with high affinity were selected and assembled to create a sequence of the linear protein. OmpC, OmpD and OmpF have previously been used as immunogenic proteins in vaccine model [Recently, Zafar et al.  reportedi et al.  and Ali i et al.  introducin (Stn) . Thus, tne model . OmpA isne model , 27. To ne model , 29. AftThe final vaccine model contains 237 amino acids with a molecular weight of 24.07 kDa which is optimum for chimeric vaccines . It was Salmonella infection due to recognition of bacterial components by TLRs, some hierarchical events initiate: bactericidal activity of macrophages, dendritic cell maturation, and production of inflammatory cytokines and chemokines. There are evidences that TLR-1, TLR2 and TLR4 play an important role in the innate immune response against NTS. Mice deficient in both TLR2 and TLR4 were highly susceptible to S. Typhimurium [In the early stages of himurium , 33.Salmonella infection suppresses humoral immunity on several levels, including B cell lymphopoiesis, MHC-II expression, germinal center development, and the survival of plasma cells [S. Typhimurium infection [The Protein–protein docking and molecular dynamics simulations of vaccine model with TLR-1, TLR2 and TLR4 revealed that the vaccine model binds to these TLRs and is probably capable of eliciting an innate and adaptive immune response. Additionally, the outcome of immune stimulation by C-ImmSim server indicates polarization of T cells response to Th1 that helped the humoral response. Previous studies indicate that ma cells , 8. In oma cells . Researcnfection . As a reE. coli expression systems are the favored host for the expression of recombinant proteins. To achieve high-level expression of the recombinant vaccine in the E. coli system, codon optimization was carried out. Since the CAI score was 1.0 and the GC content was between 30 and 60%, it is predicted that the vaccine sequence will be highly expressed in E. coli K12.Effective cloning and expression in a suitable host is a key step in the development of a multi-epitope vaccine. The S. Typhimurium and S. Enteritidis. The vaccine's antigenicity and safety were confirmed by evaluation of its toxicity, allergenicity, and solubility. Additionally, the interaction of the vaccine with TLRs shows the ability of innate immune system stimulation as well as cellular and humoral responses. This study is the first step in the vaccine production procedure and future in vivo studies will be necessary.This study collectively introduced a multi-epitope vaccine that was developed using the antigenic epitopes of the OmpA, OmpD, and enterotoxin from S. Typhimurium and S. Enteritidis, NCBI  database was chosen. Omps and Stn that have been chosen for this research include; S. Typhimurium OmpA (Accession No.: AIH09359), S. Typhimurium OmpD (Accession No.: QPE03117), S. Typhimurium Stn (Accession No.: QCQ29160), S. Enteritidis OmpA (Accession No.: UDX53918), S. Enteritidis OmpD (Accession No.: UDX71362) and S. Enteritidis Stn (Accession No.: QCQ29159). To determine the similarity between proteins in different strains of S. Typhimurium and S. Enteritidis, Omps and Stn sequences were blasted (BLAST: https://blast.ncbi.nlm.nih.gov).To retrieve Omps and Stn sequence of Salmonella infection. To predict CTL and HTL activating epitopes, IEDB (http://tools.iedb.org/main/tcell) server was used to predict peptides that bind to MHC-I and MHC-II, respectively. MHC-I epitopes were predicted based on SMM method with the most frequent alleles in the human population  [50 < 500 nM) were chosen for further analysis [The role of Cytotoxic T lymphocytes (CTL) and helper T lymphocytes (HTL) is very important in the immunogenicity of subunit vaccines particularly in dealing with alleles) . For MHCalleles) . The lenanalysis –39.https://webs.iiitd.edu.in/raghava/abcpred/ABC_submission.html) and neural network algorithm with a length of 16 amino acids and 0.51 threshold were used to predict linear B cell epitopes of OmpA, OmpD and Stn proteins [B-lymphocyte epitopes are essential for the humoral immune system and antibody production. Therefore, the ABCpred server (proteins .http://tools.iedb.org/immunogenicity) was used to determine the immunogenicity of the MHC-I epitopes [http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html) was used to assess the antigenicity of multi-epitope vaccine sequence. The server threshold was set as 0.4 for bacteria. This server utilizes the ACC (auto and cross-covariance) to produce an output [http://www.ddg-pharmfac.net/AllerTOP) was utilized [https://webs.iiitd.edu.in/raghava/toxinpred/multi_submit.php) was used to check the toxicity of all epitopes [The IEDB server  based on default consensus methods  [The MHC restriction and population coverage of anticipated epitopes for human in different regions were analyzed using the IEDB population coverage tool (Library) . This seLibrary) .https://aps.unmc.edu/database/anti). Additionally, BLASTp was utilized to evaluate the similarity of the vaccine construct with the human proteome and avoid an autoimmune response [Selected B cell and HTL epitopes were linked by GPGPG linker and CTL epitopes by AAY linker. In addition, a Griselimycin (APD ID: AP02688) was selected as an adjuvant to enhance the immunogenicity of the vaccine and bind through the EAAAK linker , 47. Theresponse .https://web.expasy.org/protparam) was used to predict the physicochemical properties of the vaccine structure including; theoretical isoelectric point (pI), half-life, molecular weight (MW), instability, aliphatic index and grand average of hydropathicity (GRAVY) (14). The Protein Sol database (https://protein-sol.manchester.ac.uk) was also used to determine the solubility of the vaccine construct. The predicted solubility is indicated by the scaled solubility value (QuerySol). Since the population average for the experimental dataset (PopAvrSol) is 0.45, any number greater than 0.45 is anticipated to have a higher solubility than the average soluble E. coli protein [The ProtParam online server  is a simple online secondary structure prediction tool that incorporates two feed-forward neural networks which predict transmembrane topology, transmembrane helix, folds, and domains efficiently [https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html) is an online tool used to indicate secondary structure–property. This method correctly predicts 69.5% of secondary structure–property  in proteins database. The combination of SOPMA and a neural networks method (PHD) predicts 82.2% of residues for 74% of co-predicted amino acids [The secondary structure of the vaccine construct was generated using PSIPRED Property online tool. PSIPRED (iciently . The SOPno acids .https://zhanglab.ccmb.med.Umich.Edu/I-TASSER) was used to create a three-dimensional (3D) structure of the vaccine construct. Based on the sequence-to-structure-to-function paradigm, I-TASSER generates three-dimensional (3D) atomic models from multiple threading alignments and iterative structural assembly simulations. The accuracy of the 3D model is provided based on the confidence score (C-score). The C-score is generally in the range between − 5 and 2, wherein a higher number indicates better quality [Iterative Threading Assembly Refinement (I-TASSER) server . This server utilizes molecular dynamics simulation to reconstruct the side chains and repack them [https://saves.mbi.ucla.edu) was used [To improve the quality of 3D structure, refinement of the vaccine model was carried out with the Galaxy Refine server  [Protein folding can put distant residues together and generate discontinuous B-cell epitopes. Since more than 90% of B cell epitopes are discontinuous, the presence of these epitopes in the vaccine structure was predicted by the Ellipro server (llipro/) . The thrllipro/) .https://cluspro.bu.edu/login.php) [https://www.rcsb.org/). For HLA-C and HLA-DRB1 these receptors docked with epitopes: ‘EAIFTPYFTE’ and ‘ALDQLYSQLSNLDPK’ respectively. To investigate the stability and physical movements of the vaccine-TLR-1, TLR-2 and TLR-4 docked complex, molecular dynamics simulation by iMODS online server (http://imods.Chaconlab.org) was performed [Since the vaccine model must first bind to immunological receptors in order to elicit an immune response. The potential of vaccine docking with immune receptors (MHC and TLR) was evaluated by ClusPro 2.0 server (gin.php) . The PDBerformed , 59. Thierformed .https://kraken.iac.rm.cnr.it/C-IMMSIM/) in order to determine the immune response profile in the in silico model. C-ImmSim server evaluates humoral and cellular response to vaccine model [The vaccine amino acid sequence was submitted to C-ImmSim server (ne model  based onhttp://www.JCat.de/) was performed for best protein expression in E. coli strain K12. The levels of protein expression are calculated based on the codon adaptation index (CAI) values (> 0.8) and GC contents (30 to 70%). Then to confirm the expression of vaccine, the optimized vaccine sequence was cloned in E. coli plasmid vector pET-30a (+) (within restriction sites of HindIII and BamHI) using the SnapGene software [Finally, the efficacy of the vaccine model in cloning and expression is important for in vitro production. To optimize vaccine construction, first, Java Codon Adaptation Tool (JCat) (software .


Leishmania infantum is the agent of visceral leishmaniasis in the Mediterranean basin. It is transmitted by sandflies of the subgenus Larroussius. Although Phlebotomus perniciosus is the most important vector in this area, an atypical Ph. perniciosus easily confused with Ph. longicuspis has been observed in North Africa. MALDI-TOF MS, an important tool for vector identification, has recently been applied for the identification of sandflies. Spectral databases presented in the literature, however, include only a limited number of Larroussius species. Our objective was to create an in-house database to identify Mediterranean sandflies and to evaluate the ability of MALDI-TOF MS to discriminate close species or atypical forms within the Larroussius subgenus. Field-caught specimens (n = 94) were identified morphologically as typical Ph. perniciosus , atypical Ph. perniciosus , Ph. longicuspis (n = 9), Ph. ariasi (n = 9), Ph. mascittii (n = 3), Ph. neglectus (n = 5), Ph. perfiliewi (n = 1), Ph. similis (n = 9) and Ph. papatasi (n = 2). Identifications were confirmed by sequencing of the mtDNA CytB region and sixteen specimens were included in the in-house database. Blind assessment on 73 specimens  showed a good agreement (98.5%) between MALDI-TOF MS and molecular identification. Discrepancies concerned confusions between Ph. perfiliewi and Ph. perniciosus. Hierarchical clustering did not allow classification of PN and PNA. The use of machine learning, however, allowed discernment between PN and PNA and between the lcus and lcx haplotypes of Ph. longicuspis . MALDI-TOF MS is a promising tool for the rapid and accurate identification of field-caught sandflies. The use of machine learning could allow to discriminate similar species. Leishmania infantum Nicolle is the main agent of visceral (VL) and cutaneous (CL) leishmaniasis. In the Mediterranean basin, the Middle East and East Africa, this zoonosis is transmitted by the bite of an infected female Phlebotomine sandfly belonging mainly to the genus Phlebotomus Rondani & Berté, subgenus Larroussius Nitzulescu [L. infantum, domestic dogs and wild foxes play the role of vertebrate hosts. Although rabbits could play the role of alternative reservoir hosts locally [tzulescu . In the  locally , the rol locally .L. infantum proven vectors are Ph. perniciosus Newtsead, Ph. longicuspis Nitzulescu, Ph. langeroni Nitzulescu, Ph. perfiliewi Parrot and Ph. ariasi Tonnoir, all belonging to the subgenus Larroussius. The most widespread and abundant species in this area and major vector is Ph. perniciosus. Ph. perfiliewi is also widespread, but with a lower frequency. The distribution areas of Ph. longicuspis, Ph. langeroni and Ph. ariasi are limited, but they can also act as vectors locally [In the Western part of the Mediterranean basin,  locally . Ph. perniciosus complex Nitzulescu [Ph. perniciosus and Ph. longicuspis. First identified in Malta, Ph. perniciosus is widely distributed throughout the western Mediterranean Basin: from Croatia to Portugal in Europe, and from Libya to Morocco in North Africa where it is predominantly found in sub-humid and semi-arid bioclimate zones [Phlebotomus longicuspis distribution is restricted to North Africa where it is most frequently recorded in semi-arid, arid, and per-arid bioclimate zones . Its identification in southern Spain [Ph. perniciosus, whereas it is simple, lightly curved and pointed in Ph. longicuspis. Moreover, Ph. perniciosus male specimens exhibit ten to 16 internal setae on their gonocoxite, whereas Ph. longicuspis exhibits 15 to 29 gonocoxal setae. The tzulescu  includeste zones . Phlebotrn Spain ,8 remainrn Spain , is onlyPh. perniciosus , with a simple and curved distal portion of parameral sheath instead of bifurcated, which can be confused with Ph. longicuspis, has been described in the western part of Mediterranean region [Ph. perniciosus, and sequence analysis of the COI fragment of mtDNA have shown a distinct haplotype for PNA [Ph. perniciosus forms, intermediate between Ph. perniciosus and Ph. longicuspis, have also been described in Spain under the Ph. longicuspis name [L. infantum VL has been reported and its absence in foci of the disease. These observations raise the question as to the role of PNA in the transmission of VL due to L. infantum. An atypical form of n region . Males on region . No morp for PNA ,12. Howe for PNA . The PNA for PNA ,15,16,17 for PNA  and Tuni for PNA ,19. Probpis name ,19. The pis name  describeMALDI-TOF MS is currently an important tool for the identification of arthropods of medical or veterinary importance . It has Ph. perniciosus complex. In 2015, Mathis et al. reported [Ph. perniciosus was noted. Discrepancies were reported for the Larroussius subgenus, particularly for a specimen identified morphologically as Ph. galilaeus whose spectrum matched with that of Ph. perniciosus. However, specimens of Ph. perniciosus did not come from areas where PNA are described.Few studies investigated field-caught sandflies belonging to the reported  on the iLarroussius and to see if this technique can distinguish between different morphotypes of Ph. perniciosus.Our objective was to evaluate the performance of MALDI-TOF MS to identify species of the genus Sandflies were collected using CDC miniature light traps  as explained in Ph. perniciosus from several countries: Portugal, Spain, France, Italy, Malta and Morocco; PNA Ph. perniciosus from Morocco, as well as a few other Larroussius . Several other species belonging to the subgenera Transphlebotomus, Phlebotomus and Paraphlebotomus were also added to our sample as out-groups  under a stereomicroscope . The head and genitalia of individual sandflies were cut off and processed for morphological analysis (see below). The body of each specimen was separated into thorax and abdomen, then stored individually in 1.5 mL sterile tubes at −20 °C.® for species identification after dehydration in successive alcoholic baths. The species were identified by observation of the head and genitalia under a BX50 microscope . Head and genitalia were processed in individual vials. Soft tissues were lysed in a bath of 10% KOH overnight, washed with distilled water, bleached in Marc-André solution overnight and mounted between a microscope slide and cover slide in EuparalGenomic DNA was extracted from the abdomen of individual sandflies using the QIAmp DNA Mini Kit  following the manufacturer’s instructions, except for crushing sandfly tissues with a piston pellet , and using an elution volume of 150 µL .v/v. Direct sequencing in both directions was performed using the primers used for DNA amplification.A fragment of 550 bp of cytochrome b (Cyt b) (using the C3B-PDR: 5′-CAYATTCAACCWGAATGATA-3′ and N1N-PDR: 5′-GGTAYWTTGCCTCGAWTTCGWTATGA-3′ primers) was amplified by PCR . AmplicoSequences obtained during the present study have been deposited in GenBank under the numbers OP617463 to OP617553 and compared to reference sequences .A phylogenetic tree was inferred using the Maximum-Likelihood method with Mega11 . The HKYThoraxes, including legs of the specimens, were placed in 10 µL of formic acid  and homogenized with a teflon pestle in a microtube. After addition of 10 µL of acetonitrile (Sigma-Aldrich) and centrifugation for 2 min at 10,000 rpm, 1 μL of supernatant was deposited on a 96-well steel target plate. Four spots were deposited for each specimen. After complete drying, 1 μL of HCCA matrix  saturated solution in organic solvent solution (Sigma-Aldrich) was added. After complete drying at room temperature, plates were then analyzed in a Bruker Microflex LT MALDI–TOF spectrometer . Spectra acquisition was repeated 8 times for each well using the built-in Bruker method “MBT_BTS_Validation_AutoX”. A bacterial test standard was used for instrument calibration. Lastly, spectra were visualized using FlexAnalysis v3.4 and imported in Biotyper Compass Explorer v4.1.100 for analysis.The Bruker “MALDI Biotyper Preprocessing Standard Method” was used for the creation of main spectra profiles (MSP). High-quality spectra were selected for incrementing the local database . Hierarchical cluster analysis (HCA) was performed using a correlation method and the Ward algorithm for clustering with the MSP dendrogram tool of Compass Explorer. Sensitivity and specificity were assessed by blind testing the in-house MSP database against spectra from 77 specimens not used for MSP creation. The cut-off value of log-score value (LSV) was determined using receptor-operated-channel curve (ROC curve) analysis with the R pROC package . Our speMachine learning discrimination between PN and PNA was based on custom R scripts based on the caret package  using foModel training was performed on the training dataset with the KNN, PLS, RF and xgbTree caret  methods,Ph. perniciosus complex was based on the observation of the top of the parameral sheath and the number of gonocoxal internal setae.Atypical Phlebotomine sandflies were clearly identified at the specific level according to their morphological characters. The unequivocal identification of the members of the Morphological identification of all specimens included in our study was confirmed by cytb mtDNA sequencing with homologies >99% versus reference sequences deposited in Genbank. A phylogenic tree inferred from the Maximum-Likelihood method is presented in Phlebotomus species using Flex analysis showed consistent and reproducible spectra for each species. Spectra of five representative specimens are presented in Ph. perfiliewi and Ph. perniciosus PN were very similar whereas Ph. perniciosus PNA displayed a different pattern of peaks intensities.Non-flat-line, usable spectra were obtained from 95 out of 99 specimens (95.95%). Visual assessment of acquired spectra from all Ph. perniciosus complex and other species. The spectra of Ph. perfiliewi (MT23) was mixed with spectra of Ph. perniciosus. For Ph. perniciosus, no clear clustering according to morphotype (PNA/PN), sex or geographical origin  was identified.MSP was obtained for 90 specimens. Cluster analysis of 64 specimens is presented in Ph. perniciosus , Ph. longicuspis lcx (2 MSP), Ph. longicuspis lcus, Ph. ariasi .High-quality spectra from 17 specimens were chosen for creating an in-house MSP library: 3496 spectra acquired from 73 specimens were tested against the upgraded homemade reference database. Determination of LSV threshold was based on molecular identification using ROC curve . The optPh similis not present in the database. The confusion matrix  reached a log score > 1.7, which was considered a probable identification at the species level. Six specimens (8.2%) displayed spectra that did not reach the LSV cut-off. One of these was MT08, a specimen of n matrix  revealedPh. perniciosus (18/19) (confused with Ph. perfiliewi (n = 13)) or Ph. tobbi (n = 5), and Ph. longicuspis (1/19) (confused with Ph. perfiliewi). The Marseille database did not allow the discrimination of lcux from lcx haplotypes of Ph. longicuspis.Spectra acquired from our 73 specimens were matched against the Marseille MSP database. 894 reached the cut-off LSV-value (25.57%). Agreement between molecular biology and MALDI-TOF was 97.87% . The 19 Ph. longicuspis were identified as Ph. perniciosus with the Reims database. Interestingly, the second identification was Ph. longicuspis in both cases.Marseille and Reims (in-house) databases were also matched against spectra from the samples of Mhaidi et al. , kindly Ph. longicuspis (lcx and lcus haplotypes), PNA and PN haplotypes of Ph. perniciosus were partitioned into (I) a training dataset (500 spectra from 53 specimens), (II) a validation dataset (124 spectra from 30 specimens) and (III) a test dataset . The training dataset was balanced concerning the two Ph. longicupis haplotypes, PN and PNA to prevent overfitting with 125 spectra for each taxa. Use of 4 common machine learning algorithms allowed to discriminate spectra from the test dataset as Ph. perniciosus PNA/PN and Ph. longicuspis lcx and lcus with good reliability  and eXtreme Gradient Boosting (XGBoost) models.Spectra from iability . The besLarrousius from non-Larroussius subgenus sandflies efficiently. Clustering according to sex, as reported by Lafri et al. [Ph. perniciosius complex, no classification errors were noted with our in-house database for the Ph. neglectus and Ph. ariasi species. Confusions between Ph. perfiliewi and Ph. perniciosus sensu stricto were, however, endured. Spectra of these closely related species were visually indistinguishable. This confusion has also been reported by other authors. In 2016, Lafri et al. [Ph. perniciosus (PN form), Ph. longicuspis and Ph. perfiliewi species of the subgenus Larroussius. They reported very similar observations between MALDI-TOF MS spectra for these two species, and a difficulty in identifying these two species using Bruker MALDI-Biotyper. MALDI-TOF MS allows to discriminate i et al. , was noti et al.  describePh. perfiliewi spectra. The remaining spectra were also classified as Ph. perniciosus. Unfortunately, only one specimen of Ph. perfiliewi was collected, thus not allowing the use of machine learning algorithms to discriminate between this two sibling species using MALDI-TOF MS. The inclusion of new specimens of Ph. perfiliewi would, however, allow the identification of Ph. perfiliewi subspecies [The Marseille database allowed the correct classification of 7 out of 14 bspecies . Both dabspecies . This rebspecies .Ph. perniciosus as previously emphasized. Pesson et coll. [Ph. perniciosus is in an intermediate position in relation to the hypothesis that its settlement could be a consequence of a late dispersion following the last glaciation [No clustering according to the haplotype was observed for PN spectra using hierarchical classification, as well as no discrimination between populations of et coll.  demonstraciation . MoreovePh. longicuspis haplotypes using MALDI-TOF MS has been obtained with machine learning. Ph. perniciosus whole genome sequencing could allow recognition of the protein origin of these discriminant peaks and could thus provide information on the genetic status of these two forms. Machine learning is an interesting approach to extract information from MALDI-TOF MS spectra. It has been used for bacteria identification, antibiotic resistance prediction [Anopheles mosquitoes [Aspergillus clones identification [Good discrimination of PN/PNA and ediction ,40,41, msquitoes  and Aspefication . It has L. infantum VL [It is important to note that the cyt b PNA (pern06) haplotype was also found in two PN males from Morocco and Tunisia . Benabdeantum VL ,20.Phlebotomus specimens, especially in the case of Ph. longicuspis and Ph. perfiliewi s. l. Unfortunately, we could not find any other specimen of the latter species in the processed collections. Hierarchical classification of spectra cannot cluster the geographical populations of Ph. perniciosus as cyt b sequences. Machine learning, however, allowed reliable discrimination between PN and PNA subspecies, Ph. longicuspis lcx and lcus haplotypes. It is a promising approach to distinguish sibling species with similar spectral profiles. The differentiation of these four taxa by morphology requires a trained microscopist and a good dissection of genitalia. The use of chaetotaxy sometimes does not permit differentiation of PNA from Ph. longicuspis when the specimen presents 15 or 16 gonocoxal setae. MALDI-TOF is a tool that does not require a formal training in entomology which can be used to quickly screen a collection of frozen sandflies. The use of MALDI-TOF in routine testing for closely related sandflies species identification remains however difficult at the present time. as methods used by laboratories worldwide need some type of standardization. Morphological expertise remains particularly important and, in many cases, identification by molecular biology remains necessary for reliable diagnosis.The collection of a large number of specimens is necessary to improve the discriminatory power of MALDI-TOF and the identifiability of


Pocillopora meandrina and Seriatopora hystrix in the South China Sea) with different life-history strategies (“competitive” and “weedy”) were targeted, and untargeted mass spectrometry metabolomics combined with molecular networking was applied to characterize their differential metabolic pathways. The results show that lyso-platelet activating factors (lyso-PAFs), diacylglyceryl carboxyhydroxymethylcholine (DGCC), aromatic amino acids, and sulfhydryl compounds were more enriched in P. meandrina, whereas new phospholipids, dehydrated phosphoglycerol dihydroceramide (de-PG DHC), monoacylglycerol (MAG), fatty acids (FA) (C < 18), short peptides, and guanidine compounds were more enriched in S. hystrix. The metabolic pathways involved immune response, energy metabolism, cellular membrane structure regulation, oxidative stress system, secondary metabolite synthesis, etc. While the immune system (lysoPAF) and secondary metabolite synthesis (aromatic amino acids and sulfhydryl compounds) facilitates fast growth and resistance to environmental stressors of P. meandrina, the cell membrane structure , energy storage (storage lipids), oxidative stress system (short peptides), and secondary metabolite synthesis (guanidine compounds) are beneficial to the survival of S. hystrix in harsh conditions. This study contributes to the understanding of the potential molecular traits underlying life-history strategies of different coral species.Life-history strategies play a critical role in susceptibility to environmental stresses for Scleractinia coral. Metabolomics, which is capable of determining the metabolic responses of biological systems to genetic and environmental changes, is competent for the characterization of species’ biological traits. In this study, two coral species ( The coral reef ecosystems possess the highest biodiversity and productivity. They provide critical marine resources  for the economic and social development of humankind. The coral community is composed of various species with different life-history strategies, and the life-history composition of coral taxa affects the capacity of the coral community to cope with climate change and human pressure . Emily S2 [8 Yuan/year [8 Yuan in 20 years since 2014 [Pocillopora meandrina, with the life-history strategy of “competitive”, is the dominant species in multiple island-reefs in the SCS [anthropogenic activities have resulted in the decrease of the LCC of P. meandrina worldwide [Seriatopora hystrix, with the life-history strategy of “weedy”, was nondominant species in all the islands in the SCS. Though S. hystrix was seldomly found on outer reef slopes, it can been found in lagoons which are generally unfavorable for coral growth in high temperatures. The previous investigation also indicated that “locally extinct” S. hystrix was found in a mesophotic coral ecosystem on Okinawa Island [P. meandrina and S. hystrix belong to the Pocilloporidae family. While these two species share some similarities in biological traits, such as branching morphology, there exist some discrepancies. While the reproductive pattern of P. meandrina is broadcast spawning, that of S. hystrix is brooding [P. meandrina is faster than that of S. hystrix [The coral reefs in the South China Sea (SCS) are located at the northern edge of the “coral triangle” in Southeast Asia. It covers an area of approximately 38,462 km2 , and accuan/year . In the uan/year , accompance 2014 . The tra the SCS ,14. It forldwide . By compa Island . In termbrooding . The gro hystrix .Montipora capitate and Pocillopora acuta by untargeted metabolomics [Life-history strategies are determined by genetic and environmental factors. Metabolomics can be regarded as the ultimate response of biological systems to genetic, transcriptomic, or environmental changes. Thus, it provides a molecular biological method for the characterization of an organism’s life-history strategy. Metabolomics has been intensively used to investigate Scleractinia coral’s response and adaptation mechanism to environmental stresses. For example, Roach et al. analyzed the metabolome difference of historically bleached and unbleached Montipora capitate . They found that the content and degree of unsaturation of diacylglyceryl carboxyhydroxymethylcholine (DGCC) were closely related to the historical bleaching phenotypes . Amanda bolomics .P. meandrina and S. hystrix in the SCS, and to analyze the potential biological functions of the differential metabolites.Mass spectrometry is one of the most commonly used methods for metabolomics analysis. It possesses the advantages of high sensitivity, label-free features, simultaneous determination of multiple metabolites, and the availability of compound identification. However, compound identification is compromised by the limited mass spectral libraries containing only a fraction of tandem mass spectra (MS/MS spectra) of endogenous metabolites. Molecular network technologies, such as global natural product social molecular networking (GNPS) and metabolite identification and dysregulated network analysis (MetDNA) , are parP. meandrina and S. hystrix were sampled from the outer reef slope and lagoon of Panshi atoll , respectively, located in the Xisha island of SCS, on 15 March 2019. The Sea Surface Temperature (SST) fluctuation was higher in the lagoon than in the outer reef slope. The monthly SSTs of the outer reef slope and lagoon in 2019 were recorded to be 28.2 ± 0.5 °C and 30.2 ± 0.3 °C respectively. The salinity, nutrient availability, and turbidity were all higher in the lagoon. In contrast, the transparency of the lagoon was noticeably lower than that of the outer reef slope. The detailed water quality parameters are referred to in the literature [P. meandrina fragments were collected in the outer reef slope of Panshi atoll and four S. hystrix fragments were collected in the lagoon. After collection, the coral fragments were transferred to −20 °C refrigeration by a bucket containing seawater for subsequent analysis.terature . In thisv/v, 7:3). The extract was centrifuged at 10,000× g for 5 min at 4 °C. The supernatant was withdrawn and stored temporarily on dry ice. The remaining coral tissue was repeatedly extracted with the method above twice and the three extracts were combined. After filtration with a 0.22 μm nylon syringe filter, the extracts were stored at −80 °C until liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. The experimental procedures are referred to in Coral tissue was washed off from the coral skeleton by a water toothpick with chilled ultrapure milli-Q water. After lyophilization, 10 mg of dried coral tissue powder containing an internal standard  was extracted by 0.5 mL of ice-cold methanol/water  and a flow rate of 200 μL/min. The percentage of mobile phase B was set as follows: 0–3 min, 5%; 3–20 min, 5–95%; 20–25 min, 95%; 25–26 min, 95–5%; 26–30 min, 5%. The injection volume of each sample was 2 µL. Data were collected in positive electrospray ionization mode with the data-dependent acquisition (DDA). A DDA method was performed by an alternating collection of full mass spectra from m/z 100 to 1000 and MS/MS spectra of the top-10 most intense compounds.The extracts were analyzed on a Thermohttp://mzmine.github.io/( accessed on 1 August 2021)). The feature extraction procedure included 11 steps: MS and MS/MS mass detection, chromatogram builder, chromatogram deconvolution, isotopic peak grouper, join aligner, feature list rows filter, peak finder, duplicate peak filter, adduct search, and fragment search. The detailed parameters for each step are provided in The chromatographic features were extracted from the raw mass spectrometry data using open-source MZmine software  for principal component analysis (PCA) and orthogonal partial least square discriminant analysis (OPLS-DA). All data were unit variance- and Pareto-scaled before PCA and OPLS-DA analysis, respectively. Variable Importance in the Projection (VIP) > 1.0 is supposed to contribute significantly to the separation of two groups. The Shapiro–Wilk test was used to test the normality of a set of data before statistical significance analysis. An independent sample https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=ae3692556f2649e1904cd23f0b04928c (accessed on 5 August 2021). The FBMN output files were visualized using Cytoscape software version 3.8.0 (https://cytoscape.org/index.html (accessed on 5 August 2021)).A mgf-formatted file and a feature quantification table exported from MZmine software were uploaded to the GNPS website for running FBMN workflow. FBMN was performed with parent and fragment mass ion tolerances of 0.02 Da, a cosine score of 0.7, minimum matched peaks of 2, library search minimum matched peaks of 2, a library search score threshold of 0.7, and a minimum peak intensity of 20,000. The feature-based molecular networking job is available at: https://www.metaboanalyst.ca/) [https://www.genome.jp/kegg/pathway.html (accessed on 5 August 2021)).The metabolic pathway analysis was performed by Metaboanalyst version 5.0 (t 2021))  and the P. meandrina and S. hystrix samples. To monitor the stability and reproducibility of instrument analysis, QC samples were injected five times in the beginning and analyzed for every three samples. A blank solution, which matched the composition of the extraction solvent, was injected before the QC sample to assess the background signal and carryover during analysis. Only metabolites detected in QC samples with a coefficient of variation (CV) < 30% were kept for further analysis. All metabolites were identified with errors within five parts per million (ppm) mass accuracy.Quality control (QC) was conducted throughout the study. QC samples were prepared by pooling an equal quantity of each coral tissue dry powder, and extracted by the same method as the P. meandrina and S. hystrix, the tissues of eight P. meandrina and four S. hystrix were parallelly extracted and analyzed by LC–high resolution mass spectrometry. The feature peaks of LC were extracted by an open source software, Mzmine. Three thousand eight hundred and fifteen metabolite feature peaks were extracted from the metabolomics dataset of P. meandrina and S. hystrix. It is noteworthy that the selected extraction protocol and mass spectrometry detection method confined the molecular coverage to polar and semi-polar compounds, because the extraction solvent of methanol/water  used in this study is inefficient for extracting non-polar and weak polar compounds, such as terpene. To maximize the ionization efficiency of extracted metabolites, an ESI source was used in mass spectrometry. To more comprehensively understand the metabolite information, the coral tissue should be multiply extracted by solvents with gradient polarities, and additional mass spectrometry instruments, including GC–MS and LC-APCI–MS, should be employed in future investigation. The signal intensities of 2044 out of the 3815 metabolites (accounting for 53.6%) were higher in P. meandrina. Correspondingly, the signal intensities of 1771 metabolites, accounting for 46.4% of the total metabolites, were higher in S. hystrix. Especially, 23 metabolites were unique to P. meandrina and 84 metabolites were unique to S. hystrix  was calculated to compare the chemical diversity of the two corals; it showed that the chemical diversity of P. meandrina was higher than S. hystrix, but there was no significant difference . To summarize, 350 metabolites were screened out as the differential metabolites between P. meandrina and S. hystrix  [P. meandrina and S. hystrix are detailed in the following.To annotate the chemical identification of metabolites, the metabolomics dataset of on (NAP)  and MetDon (NAP) , construon (NAP) . To specP. meandrina  indicated a glycerophosphoryl choline group  of lyso PC P-18:0  and m/z 522.3905 (unknown) indicated that the head group in the unknown metabolites was piperidine, compared to a quaternary ammonium group in lyso PC P-18:0  and m/z 528.344 (unknown) indicated that the head group in the unknown metabolites was pyridine (Multiple phosphatidylcholines (PC) with VIP > 1, such as lyso-platelet-activating factor (lyso-PAF) C-16, lyso-PAF C-18, lyso-PAF C-20, lyso PC P-16:0 and lyso PC P-18:0, were annotated through the spectral library searching from GNPS database. Their signal intensities were higher in eandrina . Notably hystrix . To specne group e. By comC P-18:0 f–h. For 239.0786 f, with aC P-18:0 e. Based C P-18:0 g. Since pyridine h. Nonethpyridine , which mS. hystrix  were more enriched in P. meandrina .Other lipids and FAs, including dehydrated phosphoglycerol-dihydroceramide (de-PG DHC), lyso-DGCC and monoacylglycerol (MAG), were also annotated from the molecular networking. On average, the signal intensities of de-PG DHC and MAG were higher in  hystrix , and theeandrina . Though eandrina . FurtherS. hystrix , thymidine monophosphate, 5’-methylthio-adenosine, succinyl–adenosine, propionyl–carnitine, alanopine, 1-carboxyethyl–isoleucine, S-(5’-Adenosyl)-L-homocysteine, lysine, methionine, and 4-hydroxy-D-proline, whereas S. hystrix accumulated higher levels of adenine, thymine, (4-carboxy-2-hydroxybutyl)-trimethylazanium, isovaleryl–carnitine, arginine, 4-guanidinobutyric acid, indole-3-carboxaldehyde, 3-indolepropionic acid, saccharopine, creatine, and maltose . Phenylalanine, tyrosine and tryptophan biosynthesis, and phenylalanine metabolism involved two differential aromatic amino acids . The arginine and proline metabolism involved three differential guanidyl compounds  as well as 4-hydroxy-D-proline. Cysteine and methionine metabolism involved three differential sulfhydryl metabolites -L-homocysteine). In summary, all of the five metabolic pathways belong to amino acid metabolism.The KEGG pathway enrichment analysis of the 25 differentially small molecular metabolites was performed by MetaboAnalyst 5.0 to identify the differential metabolic pathways between involved , among wMontipora capitata is preparing to spawn, it metabolizes more montiporic acids [Sarcophyton ehrenbergi changes from wild to an aquarium, it metabolizes less cembranoids [P. meandrina and S. hystrix. Their potential biological functions for the formation of the life-history strategies of P. meandrina and S. hystrix will be discussed in detail subsequently.Metabolites are the end products of cellular regulation processes, and play an important role in energy metabolism, immune regulation and oxidative stress. The abundance of metabolites can indicate the physiological status of organisms. For example, when ic acids . When thbranoids . In our P. meandrina may be beneficial to its competition with other species and rapid tissue repair after injuries, which results in the faster growth rate.Structural lipids participate in various physiological processes of organisms, such as immune regulation , construDurusdinium trenchii, contained a significantly lower amount of DGCC than thermo-sensitive Cladocopium C3 [S. hystrix was more opportunistic in accommodating temperature oscillation. For example, in the context of global climate change, the growth rate of Pocillopora genus in Lord Howe island decreased by 30% from 1994/1995 to 2010/2011. By comparison, the growth rate of S. hystrix was hardly affected [P. meandrina and S. hystrix in the SCS, P. meandrina generally lives in the outer reef slopes with strong water exchange and tiny temperature fluctuations, whereas S. hystrix was frequently found in lagoons with weak water exchange and strong temperature fluctuation in a day. In our investigation, the lower content of DGCC and lower UI of structural lipids in S. hystrix may serve to acclimate to high temperature environments by adjusting cell membrane structure. Previous investigations showed that Seriatopora caliendrum could adapt to the upwelling region with strong temperature oscillations by adjusting the content and saturation of phosphatidylcholines and plasmanylcholines [The content and degree of unsaturation of structural lipids affect the melting point of cell membranes, which further affects their physiological characteristics, such as fluidity . It is ropium C3 , and theopium C3 . Multiplaffected . Furthercholines . As for Pocillopora damicornis larvae with different life histories showed differential responses to environmental change [S. hystrix species stores more energy to promote their chances of survival in stressful environments, such as high-temperature environments. Additionally, the reproduction mode of brooding necessitates S. hystrix planula to store sufficient energy to survive long-distance dispersal [S. hystrix have a longer lifetime, which increases their chances of finding a suitable habitat before settlement [S. hystrix may facilitate the survival of both planulae and adults in unfavorable environments. On the one hand, the relatively low-concentrated MAG and relative short-chain FAs in P. meandrina may be derived from the accelerated catabolism for rapid growth. Yamashiro et al. compared the lipid content and composition of Montipora informis in Sesoko Island, Japan between normal and tumorous tissues, and they found that the tumorous coral tissue possessed a reduced level of storage lipids for the increased energy demand for tumor synthesis [The function of storage lipids, such as MAG and FA, is to provide energy for organisms. The lipid analysis of l change . While tispersal . Previouttlement . While lttlement . Therefoynthesis .P. meandrina may adapt to the environment by adjusting cell membrane structures  [S. hystrix may reinforce competitiveness with other species by strengthening its immune system  [P. meandrina and S. hystrix may acclimatize themselves to increased turbidity by storing more lipids, compensating for energy depletion due to decreased photosynthesis.In the context of global warming, it is inferred that  lipids) ,35 or in lipids) . By contyso-PAF) . Confronyso-PAF) . This enMontipora capitata and Pocillopora acuta to defend against high-temperature stress [Simplicillium sp. [S. hystrix may serve as antioxidants to resist various environmental and biological stresses in harsh conditions. Comparatively, the ideal growth environment of P. meandrina results in the lower production of small peptides.Coral hosts or symbiotic microorganisms can synthesize peptides with a variety of biological functions, including anti-inflammatory/antioxidant , antibace stress . Cyclo in organisms [P. meandrina might reduce oxidative damage under adverse conditions, such as storm damage and excessive ultraviolet radiation. Guanidyl compounds are important nitrogen sources and relate to the microbial community composition [S. hystrix may favor its survival in nitrogen-deficient environments [Aromatic amino acids, sulfhydryl compounds, and guanidyl compounds jointly participate in the regulation of the life-history traits of ynthesis ,50. Aromynthesis , oxidatiynthesis , and parynthesis . Previouynthesis . Thereforganisms . The eleposition ,56. The ronments . With thP. meandrina and weedy S. hystrix. While lyso-PAF and DGCC were more enriched in P. meandrina, new phospholipids, de-PG DHC, MAG and FAs (C < 18) were more enriched in S. hystrix  and biotic  stresses, and to explore the differential tolerance mechanism of the same coral species from different growing habitats to environmental change, providing theoretical basis for screening resistant corals.


Actinobacillus pleuropneumoniae and Pasteurella multocida are two important bacterial pathogens in swine industry. In the present study, resistance profiles of nine commonly used antibiotics of A. pleuropneumoniae and P. multocida isolates of swine origin from different regions of China were investigated by determination of minimum inhibitory concentrations (MICs). In addition, genetic relationship of the florfenicol-resistant A. pleuropneumoniae and P. multocida isolates was determined by pulsed-field gel electrophoresis (PFGE). The genetic basis of florfenicol resistance in these isolates were explored by floR detection and whole genome sequencing. High resistance rates (>25%) of florfenicol, tetracycline and trimethoprim- sulfamethoxazole were observed for both bacteria. No ceftiofur- and tiamulin- resistant isolates were detected. Furthermore, all the 17 florfenicol-resistant isolates (nine for A. pleuropneumoniae and eight for P. multocida) were positive for floR gene. The presence of similar PFGE types in these isolates suggested that clonal expansion of some floR-producing strains occurred in the pig farms from same regions. WGS and PCR screening showed that three plasmids, named pFA11, pMAF5, and pMAF6, were the cargos of the floR genes in the 17 isolates. Plasmid pFA11 exhibited novel structure and carried several resistance genes, including floR, sul2, aacC2d, strA, strB, and blaROB − 1. Plasmids pMAF5 and pMAF6 were presented in A. pleuropneumoniae and P. multocida isolates from different regions, suggesting horizontal transfer of the two plasmids are important for the floR dissemination in these Pasteurellaceae pathogens. Further studies of florfenicol resistance and its transfer vectors in Pasteurellaceae bacteria of veterinary origin are warranted. Actinobacillus pleuropneumoniae and Pasteurella multocida, two members of the family Pasteurellaceae, are important pathogens and cause significant economic losses in swine industry worldwide  and eight LPS genotypes (L1–L8)  . Of them . Of thefections . For thihe world .A. pleuropneumoniae and P. multocida infections. Florfenicol-resistant A. pleuropneumoniae and P. multocida isolates have been sporadically reported in different countries and, same to other Gram-negative bacteria, floR is the predominant determinant for resistance to florfenicol in those isolates (floR was mainly carried by plasmids with various sizes or integrative conjugative elements (ICEs)  , 15–21.A. pleuropneumoniae and P. multocida isolates from swine in China. In the present study, we collected A. pleuropneumoniae and P. multocida isolates from pig farms in different regions of China, and characterized the genetic relationships of the florfenicol-resistant isolates and the structures of relevant floR-carrying plasmids.There are few data about the florfenicol resistance and its genetic basis in A. pleuropneumoniae isolates and 31 P. multocida isolates were used in this study. These isolates were collected between September 2018 to December 2020 in routine clinical diagnosis in several pig farms from different provinces of China, including Jiangsu, Xinjiang, Shanghai and Hubei. Minimum inhibitory concentrations (MICs) were determined by broth microdilution method according to recommendation of the Clinical and Laboratory Standards Institute (CLSI) supplement VET08  value given by European Committee on Antimicrobial Susceptibility Testing (EUCAST) was temporarily used as resistance breakpoint (https://www.eucast.org/). In addition, CLSI has no clinical breakpoints of tiamulin, gentamicin, and trimethoprim-sulfamethoxazole for P. multocida and the ECOFF values from EUCAST were also used as resistance breakpoints for these antibiotics. Moreover, MIC50 and MIC90 were exhibited to reflect the MIC distributions. Escherichia coli ATCC 25922 and A. pleuropneumoniae ATCC 27090 were used as quality control strains.A total of 19 nt VET08 . Nine cofloR gene with specific primer sets (5′-CTGAACACGACGCCCGCTATG-3′ and 5′-CAGGACCGCTCCGCAAACAA-3′) with the amplification condition: 95°C for 5 min; followed by 30 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 60 s; and extension at 72°C for 7 min. Sanger sequencing of the amplicons was performed to confirm the PCR results. To investigate the genetic relationship between the floR-positive isolates, ApaI-PFGE was performed as described previously with modified running parameter: 14°C, 6 V/cm, 2.16 s initial switching time, 54.2 s final switching time and a 18 h of total run  (www.ncbi.nlm.nih.gov/BLAST) . PCRs with the three primer sets were conducted to detect the plasmid types in the floR-producing isolates without WGS analysis. The amplicons were sequenced and compared with corresponding plasmids to confirm the PCR results.The genetically unrelated dr.org/) . Structuv/BLAST) . MoreovefloR-carrying plasmids pFA11, pMAF5 and pMAF6 have been deposited in the GenBank database with the accession numbers CP100665, CP100664, CP100663.The complete sequences of the A. pleuropneumoniae and 31 P. multocida isolates were exhibited in A. pleuropneumoniae isolates, high resistance rates were observed for tetracycline and trimethoprim-sulfamethoxazole , while florfenicol showed moderate degree of resistance (47.4%). Low frequencies of resistance were observed for ampicillin, enrofloxacin, tilmicosin and gentamicin . No resistance of ceftiofur and tiamulin were detected for these A. pleuropneumoniae isolates. In P. multocida isolates, highest resistance rate was observed for trimethoprim-sulfamethoxazole (87.1%). Eight and twelve isolates showed resistance to florfenicol and tetracycline, corresponding to resistance rates 25.8 and 38.7%, respectively. For other test antibiotics, no resistance was detected.Resistance profiles of the 19 Enterobacteriaceae, resistance to florfenicol in A. pleuropneumoniae and swine-origin P. multocida isolates has rarely been reported all over the world . All of these strains were positive for floR gene. These data indicated that floR-mediated florfenicol resistance in P. multocida and A. pleuropneumoniae isolates have emerged and spread in pig farms of China in recent years. According to our survey, florfenicol is one of the most frequently applied antibiotics in the pig farms where the P. multocida and A. pleuropneumoniae strains used in this study were recovered. The high selection pressure due to extensive use of florfenicol in these farms may promote the dissemination of floR gene and associated resistant isolates. The genetic relationship of these floR-producing isolates was investigated by PFGE . Besides floR, pFA11 also contained sulfonamide resistance gene sul2, aminoglycoside resistance genes aacC2d, strA, strB, and β-lactam resistance gene blaROB − 1. Comparison with sequences deposited in Genbank showed that pFA11 shared highly structural similarity with plasmids originated from other Pasteurellaceae bacteria such as pIMD50 from A. porcitonsillarum of swine origin in Switzerland (GenBank accession no. AJ830711) and pOV described in P. multocida isolated from Mexico (GenBank accession no. JX827416)   32, 33), 33A. plrep, a resistance gene floR and four putative ORFs were present. Database searching showed that pMAF5 was highly similar to plasmid pHPSGC from G. parasuis HpsGC of sick pig in China (GenBank accession no. KX966395) (floR and its flanking region (lysR-floR-hp) showed high homology to the corresponding region of plasmid pCCK381 described in P. multocida isolate strain 381 from lung of a calf origin from UK (GenBank accession no. AJ871969) , p780 originated from A. pleuropneumoniae MV780 in Brazil (GenBank accession no. MH457196) and pM3362MDR from A. pleuropneumoniae MID3362 originated from pig lung in UK (GenBank accession no. MH457202) (tet (B), blaROB − 1, sul2 and strA, but not floR.pMAF5 was 5,279 bp in length with simple structure, in which a plasmid replication gene X966395) . StructuJ871969) . The repH457202) . These pfloR-vector, which was detected in eight isolates from different regions and with various PFGE patterns. pMAF6 was 6, 349 bp in size and showed >99% nucleotide identity to previously reported floR-bearing plasmid pHPSF1 identified from G. parasuis ASB17W in China (GenBank accession no. KR262062) . The same plasmid structure was also observed for plasmid p11745 in A. pleuropneumoniae APP11745 from Spain (GenBank accession no. DQ176855), which served as the cargo of tetracycline resistance gene tet (B)    . Compare tet (B) . MoreoveA. pleuropneumoniae and P. multocida isolates of swine origin in China was analyzed in this study. Considering the importance of florfenicol in pig industry, the high prevalence of florfenicol resistance and floR gene observed in this study may significantly affect the control of A. pleuropneumoniae and P. multocida infections in swine respiratory diseases. Moreover, the prevalence of small plasmids with similar structure in various isolates of Pasteurellaceae bacteria from different regions revealed that this kind of plasmids are important cargos for the transmission of not only floR but also other resistance genes in porcine Pasteurellaceae pathogens. Continuous monitoring of florfenicol resistance and prevalence of floR gene and associated plasmids in Pasteurellaceae pathogens of veterinary origin are warranted, as this information is important for the control of infections caused by pathogenic species of Pasteurellaceae in food-producing animals.The genetic basis of the florfenicol resistance in The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.Ethical review and approval was not required for the animal study because the study was performed with resistant bacteria. Samples were taken by authorized veterinarians working in the farms. The researchers did not perform any animal studies.BL and LX designed and supervised the study. WZ, YQ, KL, JW, ZL, QS, and ZM provided resources for this study. XY and QS laboratory analysis and statistical analysis. BL and XY wrote the manuscript. All authors contributed to this article and approved the submitted version.


Staphylococcus aureus bloodstream infections, but racial, ethnic, and socioeconomic disparities associated with this outcome are not well described.Racial and ethnic minorities are disproportionately affected by end-stage kidney disease (ESKD). ESKD patients on dialysis are at increased risk for Surveillance data from the 2020 National Healthcare Safety Network (NHSN) and the 2017–2020 Emerging Infections Program (EIP) were used to describe bloodstream infections among patients on hemodialysis  and were linked to population-based data sources  to examine associations with race, ethnicity, and social determinants of health.S. aureus. Among seven EIP sites, the S. aureus bloodstream infection rate during 2017–2020 was 100 times higher among hemodialysis patients  than among adults not on hemodialysis . Unadjusted S. aureus bloodstream infection rates were highest among non-Hispanic Black or African American (Black) and Hispanic or Latino (Hispanic) hemodialysis patients. Vascular access via central venous catheter was strongly associated with S. aureus bloodstream infections . Adjusting for EIP site of residence, sex, and vascular access type, S. aureus bloodstream infection risk in EIP was highest in Hispanic patients , and patients aged 18–49 years . Areas with higher poverty levels, crowding, and lower education levels accounted for disproportionately higher proportions of hemodialysis-associated S. aureus bloodstream infections.In 2020, 4,840 dialysis facilities reported 14,822 bloodstream infections to NHSN; 34.2% were attributable to S. aureus infections. Health care providers and public health professionals should prioritize prevention and optimized treatment of ESKD, identify and address barriers to lower-risk vascular access placement, and implement established best practices to prevent bloodstream infections.Disparities exist in hemodialysis-associated  A statistical modelS. aureus bloodstream infection incidence with patient vascular access type and selected dialysis facility characteristics, including those related to infection control practices (45 variables), and SVI data (20 variables)  from CDC’s EIP surveillance in selected counties from seven sites (selected counties within each state) during 2017–2020 were analyzed; data on race and ethnicity were included.S. aureus bloodstream infection rates were calculated for hemodialysis patients and the general population  for the surveillance area. EIP S. aureus bloodstream infection data were used as the numerator for calculating rates. The denominators for S. aureus bloodstream infection rates among hemodialysis patients were obtained as follows: for each year of data, aggregated county-level denominator data on the number of hemodialysis patients  were obtained from USRDS. USRDS provided the data stratified by sex, age group , race and ethnicity, and vascular access type  for each year. The denominator for calculating S. aureus bloodstream infection rates among the general population was obtained by subtracting the hemodialysis population from the total population of the surveillance area. Population totals for the entire surveillance catchment area  were obtained from U.S. Census Bureau data.***Unadjusted S. aureus bloodstream infection rates among hemodialysis patients were stratified by the characteristics described in the USRDS data. To handle overdispersion, negative binomial regression was performed to determine aRRs for age, race and ethnicity, sex, vascular access type, and EIP site.Unadjusted NHSN. During 2020, 4,840 dialysis facilities  reported 14,822 bloodstream infections; S. aureus was isolated from 5,070 (34.2%), yielding a rate of 0.1 S. aureus bloodstream infections per 100 patient-months. Among reported S. aureus bloodstream infections, 2,602 (51.3%) were identified as methicillin-sensitive and 1,923 (37.9%) as MRSA; 545 (10.7%) had no susceptibility test results reported.S. aureus bloodstream infection incidence indicated that S. aureus bloodstream infection risk was most strongly associated with patient vascular access type: compared with fistula access, CVC and graft or other had approximately six times  and approximately two times  higher risk, respectively , not being part of a chain of dialysis centers , not having a written antibiotic use policy , and location of the facility in an area with a higher proportion of persons aged ≥65 years   .EIP. During 2017–2020, 2,800 S. aureus bloodstream infections among hemodialysis patients were reported in the EIP surveillance areas. The overall annual S. aureus bloodstream infection rate was 100 times higher in hemodialysis patients  than among adults not on hemodialysis . U.S. Census Bureau tracts with lower SES factors accounted for disproportionately higher proportions of hemodialysis-associated S. aureus bloodstream infections , S. aureus bloodstream infection rates observed in Black and Hispanic patients support the higher infection risk described in other published reports , and patients on dialysis are at increased risk for S. aureus bloodstream infections, race, ethnicity, and socioeconomic factors also affected infection rates and distribution, with Hispanic or Latino ethnicity as an independent risk factor.Although vascular access type was the major risk factor for hemodialysis-associated Health care providers should prioritize prevention and optimized treatment of ESKD, identify and address barriers to lower-risk vascular access placement, and implement established best practices to prevent bloodstream infections.


This article  includesAnn Hum Biol. 2017; 44(2):164–174. (https://doi.org/10.1080/03014460.2016.1207805) PMID: 27356680Gurkan C, Sevay H, Demirdov DK, Hossoz S, Ceker D, Terali K, Erol AS. Turkish Cypriot paternal lineages bear an autochthonous character and closest resemblance to those from neighboring Near Eastern populations. In addition, the article does not include accurate terminology when describing the Syriac samples obtained from refugee camps in Erbil, Iraq. These samples were described in  as SyriaThis pertains to the following in-text citations, summarized below in Results derived from analyses of the Erbil camp samples cannot be generalized as representing findings about the Syrian population.PLOS ONE for these errors in the published article [The authors apologize to the authors of , the Edi article .


Elizabethkingia spp. is a ubiquitous pathogenic bacterium that has been identified as the causal agent for a variety of conditions such as meningitis, pneumonia, necrotizing fasciitis, endophthalmitis, and sepsis and is emerging as a global threat including in Southeast Asia. Elizabethkingia infections tend to be associated with high mortality rates (18.2–41%) and are mostly observed in neonates and immunocompromised patients. Difficulties in precisely identifying Elizabethkingia at the species level by traditional methods have hampered our understanding of this genus in human infections. In Southeast Asian countries, hospital outbreaks have usually been ascribed to E. meningoseptica, whereas in Singapore, E. anophelis was reported as the main Elizabethkingia spp. associated with hospital settings. Misidentification of Elizabethkingia spp. could, however, underestimate the number of cases attributed to the bacterium, as precise identification requires tools such as MALDI-TOF MS, and particularly whole-genome sequencing, which are not available in most hospital laboratories. Elizabethkingia spp. has an unusual antibiotic resistance pattern for a Gram-negative bacterium with a limited number of horizontal gene transfers, which suggests an intrinsic origin for its multidrug resistance. Efforts to prevent and further understand Elizabethkingia spp. infections and limit its spread must rise to this new challenge. Elizabethkingia have recently emerged as an important pathogen in hospital-acquired infections and are generally associated with high mortality [Elizabethkingia meningoseptica infections have also been associated with COVID-19 patients [Elizabethkingia spp. infects not only immunocompromised patients but also immunocompetent ones [The Gram-negative bacteria of the genus ortality . Recent ortality , accompaortality ,4, osteoortality , urinaryortality ,7, endogortality , endocarortality , epididyortality , pulmonaortality , necrotiortality ,13, cystortality , hydroceortality , and secortality . Elizabepatients . Elizabeent ones ,19,20.Elizabethkingia was that of 19 cases of meningitis in infants in the United States of America [E. meningoseptica was reported among neonates in the Congo [Historically, the first report of human infection due to  America . Even inhe Congo  with varE. meningoseptica were reportedly high amongst immunocompetent neonates as well as hospitalized patients with existing underlying infections, and in a comprehensive review, Dzuiban et al. [Elizabethkingia spp. infections, the isolation of the pathogen from the environment [Elizabethkingia spp. infections in most countries around the region. The aim of this review is to summarize our current understanding of the characteristics of Elizabethkingia spp., the current epidemiological developments, and clinical manifestations of Elizabethkingia spp. in Southeast Asia.Worldwide infections caused by n et al.  showed tn et al. . Infectin et al. , Singapon et al. , Thailann et al. , Indonesn et al. , and Camn et al. . Howeverironment ,28 suggeFlavobacterium meningosepticum, which was later recommended to be changed to Chryseobacterium meningosepticum (in 1994) [Elizabethkingia  under the Flavobacteriaceae family based on 16S rRNA phylogenetic studies [Elizabethkingia into eight species, namely E. meningoseptica, E. miricola, E. anophelis, E. bruuniana, E. ursingii, E. occulta [E. argenteiflava sp. nov. [E. umeracha [When first discovered in 1959, the suggested name for the bacterium was in 1994) . In 2005 studies . Recentl occulta , E. argesp. nov. , and theumeracha .Elizabethkingia is difficult using traditional microbiological methods and misidentification of E. anophelis with E. meningoseptica has been found to be common , it is therefore highly likely for this pathogen to be underreported. Correct identification of the organism is crucial for the diagnosis and management strategies, as E. anophelis is a nososcomial pathogen [E. anophelis and E. meningoseptica requires accurate microbial identification, but the phenotypic similarities between E. anophelis and E. meningoseptica present a challenge to accurate identification, particularly for clinically derived isolates; 16S rRNA gene analysis had identified a 98.6% similarity between E. meningoseptica and E. anophelis, which has often led to the misidentification of these bacteria [Since correct identification of pathogen . Hence, bacteria .Elizabethkingia in their reference spectra database. Studies have also shown that misidentification of Elizabethkingia was rife using these automated identification systems, with E. anophelis commonly misidentified as E. meningoseptica [Elizabethkingia, clinical isolate identifications were compared with 16S rRNA gene sequencing; it was reported that species identification concordance between these identification systems and 16S rRNA gene sequencing was low at only 24.5–26.5% [E. meningoseptica from E. anophelis, but not the remaining species of the genus Elizabethkingia [Elizabethkingia, with a solitary isolate of E. bruuniana being the only species that was misidentified. Whole-genome sequencing confirmed that the majority of the isolates were E. anophelis (n = 22), with the rest being E. miricola (n = 3), E. meningoseptica (n = 2), and E. bruuniana (n = 1) [The four automated bacterial identification systems that are commonly used in diagnostic laboratories are: 1) API/ID32 Phenotyping Kits ; (2) Phoenix 100 ID/AST Automated Microbiology System ; (3) VITEK 2 Automated Identification System ; and (4) API/ID32oseptica ,37. When.5–26.5% . Neverththkingia . In a re (n = 1) .Elizabethkingia species spectra into the databases should ensure highly accurate identification using MALDI-TOF MS systems, making it a reliable identification tool in lieu of whole-genome sequencing.In the near future, the inclusion of novel Elizabethkingia are intrinsically resistant to most β-lactams, β-lactam/lactamase inhibitors, and carbapenems due to the presence of two unique class B metallo-β-lactamases (MBLs), namely blaBlaB and blaGOB, along with a class A extended-spectrum β-lactamase (ESBL), blaCME [Elizabethkingia are the only known bacteria thus far with multiple chromosomally encoded MBLs [blaBlaB-1 [blaBlaB, and blaGOB in both E. meningoseptica and E. anophelis [blaBlaB-16 and blaGOB-19 in E. miricola isolated from a black-spotted frog in China [Elizabethkingia spp. a possible environmental reservoir for β-lactam resistance., blaCME ,40,41. Eded MBLs . Reportsin China , make ElElizabethkingia isolates are frequently resistant to aminoglycosides, macrolides, tetracycline, and vancomycin but show variable susceptibility to piperacillin, piperacillin-tazobactam, fluoroquinolones, minocycline, tigecycline, and trimethoprim-sulfamethoxazole [Elizabethkingia, and susceptibilities are largely reported based on Enterobacteriaceae breakpoints of the Clinical and Laboratory Standards Institute (CLSI) M100 guidelines and/or the European Committee on Antimicrobial Susceptibility Testing (EUCAST) pharmacokinetic–pharmacodynamic (PK–PD) “non-species” breakpoints [Elizabethkingia, and broth microdilution is instead recommended for susceptibility determination [E. meningoseptica isolate in the Kuala Lumpur General Hospital, which developed resistance during treatment to cefepime, a cephalosporin antibiotic that is normally highly active against both Gram-positive and Gram-negative organisms [hoxazole ,44,45,46hoxazole ,48,49, choxazole ; and levhoxazole . There aakpoints ,38. It hmination . Althougmination . A similrganisms .Elizabethkingia isolates from Hong Kong as susceptible to vancomycin. However, studies using broth microdilution tests on isolates from Taiwan [Elizabethkingia isolates from South Korea using the agar dilution method and concluded that all isolates were non-susceptible based on the interpretive criteria used for Staphylococcus spp. The vancomycin resistance gene, vanW, was reported in the majority of Elizabethkingia genomes, although the exact function of vanW is currently unknown [vanW have been identified in microorganisms with VanB-type glycopeptide resistance [Elizabethkingia infections [Elizabethkingia meningitis, ciprofloxacin, linezolid, or rifampicin should also be included until future clinical studies could be carried out to conclusively determine the clinical efficacy of these vancomycin-combination regiments for treatment [Using disk diffusion, Lau, Chow  reportedm Taiwan ,55 and Am Taiwan  indicatem Taiwan , who inv unknown ,46. Howesistance . In viewfections ,58, it wreatment .Elizabethkingia spp. strains from Southeast Asia was from Singapore, whereby sputum isolates obtained from three patients  and four from the hospital’s sink  at the National University Hospital, Singapore, were compared against five previously sequenced E. anophelis strains Ag1 (PRJNA80705) and R26 (PRJNA178189), E. meningoseptica ATCC 12535 (NITE) (PRJNA199489), E. meningoseptica ATCC 12535 (OSU) (PRJNA198814), and E. meningoseptica 502 (PRJNA176121). This led to the discovery of 16 antibiotic resistance genes from the core genomes and 19 antibiotic resistance genes from the accessory genomes of Elizabethkingia spp., and this included genes that confer resistance to aminoglycosides, β-lactams, fluoroquinolones, glycopeptides, macrolide-lincosamide-streptogramins, tetracyclines, trimethoprim, and rifampicin [Elizabethkingia from many different regions, including Malaysian and Singaporean genomic sequences that were available at that time, identified that all Elizabethkingia genomes contained at least 17 antimicrobial resistance genes  was used to predict their presence from the genome sequences of various Elizabethkingia spp., this led to the prediction of a total of 270 putative virulence factor genes. More than fourteen virulence factor classes for Elizabethkingia spp. were identified (see Elizabethkingia shared the same virulence factors (The mechanisms of pathogenesis of  studied . When th factors .E. anophelis (E. meningoseptica carried six unique genes involved in adherence that encode curli nucleator protein (csgB), curli assembly proteins , a curli production assembly protein (csgG), and two genes involved in immune modulation encoding a capsular polysaccharide synthesis enzyme (cap8O), a gene encoding Rab2-interacting conserved protein A (ricA) and a putative carbonic anhydrase-encoded gene (mig-5) (E. miricola unique virulence genes were predicted to be involved with urease accessory protein (ureE), urease alpha subunit (ureA), twitching motility protein (pilG), and sphingomyelinase-c (smcL)  . Four ofc (smcL) .E. anophelis highlighted the genomic heterogeneity of Elizabethkingia species [E. anopheles were first reported by Breurec, Criscuolo [cps clusters observed amongst the different lineages suggesting virulence heterogeneity among Elizabethkingia strains [capD [adeG gene for the AdeFGH efflux pump [Elizabethkingia species , catalase/(hydro)peroxidase (katA), 60K heat shock protein (htpB), phospholipase C (plc), phosphopyruvate hydratase (eno), translation elongation factor (tuf), catalase/peroxidase HPI (katG), and aspartate 1-decarboxylase precursor (panD), which is involved with adherence, biofilm formation, cellular metabolism, exotoxin production, and stress survival (icl) plays an important role in the glyoxylate cycle [Elizabethkingia spp. can predict its essential role in stationary-phase survival. An early report had shown that the presence of icl in Mycobacterium tuberculosis promoted the tenacity of infection by helping the pathogen to survive inside macrophages [Nine virulence factor genes were shared between six of the survival . Isocitrte cycle , and itsrophages .plc) in all Elizabethkingia spp. [L. monocytogenes, plc aided bacterial escape toward the cytosol and cell-to-cell propagation, whereas, in C. perfringens, it helped bacteria induce endothelial damage and platelet aggregation, and in P. aeruginosa, it led to the triggering of signaling pathways that lead to inflammation [However, the specific role of bacterial enzymes in pathogenesis varies with infection. The presence of phospholipases C (gia spp.  suggest gia spp. . In L. mammation . katA and katG (encoding hydroperoxidase I), are crucial against oxidative stress [katA were resistant to dodecyl sulfate, proteinase K, pepsin, trypsin, chymotrypsin, and the neutrophil protease cathepsin G, and they could survive for a long period once released from lysed cells [katA [katG [Elizabethkingia species’ resistance to aminoglycosides.The catalase-peroxidase genes, e stress . An earled cells . Presencls [katA ,61,72,73tA [katG ,46,72,73Elizabethkingia are aerobic, non-fermenting, non-motile, catalase-positive, oxidase-positive, indole-positive, and Gram-negative bacilli widely distributed in soil, mosquitoes, plants, fresh and marine fish [The genera ine fish ,75, foodine fish , hospitaine fish , stagnanine fish . Due to ine fish , they haine fish   suffering from red leg syndrome and cataract in Sabah, Malaysia [E. meningoseptica from bullfrogs was also described in an earlier study, in which the isolates obtained were found to be resistant to multiple antibiotics [E. miricola, which had been implicated in acute infections in humans, caused a disease outbreak associated involving the internal organs of different anuran species, including northern leopard frogs (Lithobates pipiens), Chapa bug-eyed frogs (Theloderma bicolor), and Vietnamese warty toads (Bombina microdeladigitora) captured in Vietnam. The presence of β-lactamases and putative virulence genes in the E. miricola isolates were detected in silico [Zainuri et al. (2013) reported on the isolation of Malaysia . Isolatiibiotics . E. mirin silico .E. miricola was also reportedly isolated from Tra catfish  fillets in the industrial processing lines in Vietnam [E. meningoseptica from retail sausages in Kampar, Malaysia, although the identification was performed by traditional biochemical methods and identified as Chryseobacterium meningosepticum [ Vietnam . Tra cat Vietnam . Other ssepticum .Gnetum gnemon and nearby bulk soils of a tropical forest arboretum at the Forest Research Institute of Malaysia (FRIM) at Kepong, near Kuala Lumpur, identified the mutualistic presence of E. meningoseptica and E. miricola [Elizabethkingia spp. was surprisingly found in relative abundance (4.9%) on the leaves of Gnetum gnemon in comparison with rhizoplane (1.4%) [Elizabethkingia spp. in the environment and, thus, the difficulty in tracing an outbreak should one occur in the community and outside of hospital settings.Furthermore, 454 pyrosequencing of the 16S rRNA gene from the bacterial community of the root of the gnetalean gymnosperm miricola . Elizabee (1.4%) . These rElizabethkingia spp. infections in Malaysia occurred as isolated cases rather than outbreaks, and the most dominant strain is E. meningoseptica  by the University of Malaya but as E. meningoseptica. This led a group of scientists from the University of Malaya to revive thirty archived lyophilized isolates collected from their University Hospital that were initially identified as Flavobacterium meningosepticum [E. anophelis, whereas the remaining six were E. miricola [E. meningoseptica, underlining the very high possibility of misidentification of these pathogens from earlier publications, particularly those that relied on traditional biochemical tests for their identification. Hence, the cases that had been previously reported as infections due to E. meningoseptica and reviewed below should be taken with caution.Most reported cases of oseptica . These esepticum . Re-idenmiricola . None ofE. meningoseptica (then reported as Flavobacterium meningosepticum) infection that was recorded in Malaysia involved three neonates with meningitis, where the infection was rapidly controlled by the use of rifamycin [E. meningoseptica (then reported as Flavobacterium meningosepticum) collected over a two-year period from neonates with meningitis in Kuala Lumpur [Flavobacterium meningosepticum isolates in infants from the cerebrospinal fluid, three from the blood, and one from the peritoneal fluid [E. meningoseptica infection and were treated with rifampicin, erythromycin, and novobiocin [The first case of ifamycin . Anothera Lumpur . From 19al fluid . Out of vobiocin .E. meningoseptica was confirmed at HKL and was successfully treated with the fourth-generation cephalosporin, cefepime [E. meningoseptica isolate in a bilateral simultaneous hypopyon corneal ulcer in a contact lens wearer caused by polymicrobial infection [E. meningoseptica [E. meningoseptica [E. meningoseptica in Malaysia was reported in a hemorrhagic stroke patient who developed septic shock during prolonged neuro-intensive care management at HUSM [Another case study of resistant cefepime . Ali andnfection . In addioseptica . In anotoseptica . Septice at HUSM .E. meningoseptica. Most of the isolates were from the surgical wards. Another study [E. meningoseptica from the blood and cerebrospinal fluid (CSF) of a premature infant of a dichorionic diamniotic (DCDA) twin with neonatal meningitis. The infant required non-invasive continuous pressure ventilation and, after 10 days, was noted to be febrile, less active, and developed seizures. The patient was successfully treated with intravenous vancomycin and ciprofloxacin for 6 weeks, and oral rifampicin was given for a total of 8 days according to the susceptibility testing of the organism. However, the patient’s recovery was complicated with hydrocephalus.Findings from a study conducted at a 562-bed tertiary hospital in Selangor, Malaysia , showed er study  reportedE. meningoseptica in one of the cases, which was classified as a “non-tuberculosis case” using standard case definitions.A prospective cohort study was conducted on gastrointestinal tuberculosis-suspected patients at the Queen Elizabeth Adult Hospital in Kota Kinabalu, Sabah, Malaysia . InteresE. anophelis had been reported as the dominant species of Elizabethkingia in Singapore [E. meningoseptica. Between April and June 2011, the National University Hospital (NUH) Singapore isolated three imipinem-resistant E. meningoseptica from rectal swabs of patients [E. meningoseptica in ICUs after an environmental sampling control. About 44% (35/79) of the collected tap water samples were positive with E. meningoseptica [Although ingapore , it is npatients . Anotheroseptica .E. meningoseptica infection as identified by MALDI-TOF MS [E. anophelis strains that were isolated from the midgut of the Anopheles gambiae malaria mosquito vector than to E. meningoseptica [E. anophelis outbreak in an ICU in Singapore.During a three-week period in 2012, an investigation of an outbreak by the hospital infection control team at NUH showed that three cardiothoracic ICU patients and two surgical ICU patients were initially diagnosed with I-TOF MS . All patoseptica ,115, thuE. meningoseptica in all her four blood cultures after spending 2 months in the hospital for treatment of other conditions [E. meningoseptica. The patient was injected with intravitreal vancomycin and amikacin and had started on hourly fortified gentamicin and cefazolin eyedrops. The patient’s vitreous culture results showed that E. meningoseptica was susceptible to several antibiotics, including ciprofloxacin, levofloxacin, cotrimoxazole, and minocycline, but nevertheless resistant to ceftazidime, gentamicin, and amikacin. Thus, the intravitreal administration was switched to ciprofloxacin (100 µg/0.05 mL) and repeated five times. The patient’s anterior chamber fibrin clot progressively resolved and the inflammatory material in the vitreous cavity became organized, whereas vision was not recovered [E. meningoseptica after 30 days of hospitalization at Tan Tock Seng Hospital (TTSH), Singapore [Another case involved a 75-year-old patient with numerous comorbidities diagnosed with nditions . Three decovered . Anotheringapore .E. meningoseptica using MALDI-TOF MS (VITEK MS) within 13 days in the ICU at Kandang Kerbau Women’s and Children’s Hospital (KKH), Singapore in 2017 [E. anophelis, and 1 sample was positive with E. meningoseptica. E. meningoseptica was isolated from a water tap not associated with any of the cases, and E. anophelis was isolated from an aerator [Three patients aged 2.8 months, 4.9 months, and 4.8 years were diagnosed with  in 2017 . Further aerator .E. meningoseptica (96.2%) or E. miricola (3.8%). Further 16S rRNA gene sequencing using universal primers was performed, and 77 samples showed closer nucleotide identity to E. anophelis. One sample each had a closer nucleotide identity to E. anophelis subsp. Endophytica and E. meningoseptica [Elizabethkingia species, many isolates were initially misidentified as E. meningoseptica . Among the 77 isolates collected from hospital waste matters, the blaSHV-producing E. meningoseptica strain was identified among the most predominant taxa (1.4%), showing resistance to extended-spectrum cephalosporins and carbapenems [In another retrospective study from 2009 to May 2017 conducted by Chew et al. (2018), seventy-nine blood isolates analyzed with Bruker MALDI Biotyper (bioMérieux) resulted in the identification of either oseptica . Due to bapenems .E. meningoseptica infection from the blood and 4 from CSF samples [E. meningoseptica bacteremia. However, a high (75%) number of morbidities consist of patients presented with meningitis. Most patients had developed post-infectious conditions such as hydrocephalus, quadriplegic cerebral palsy with severe disability, and reduced academic performance, whereas another had moderate global developmental delay.An eight-year retrospective descriptive study (2010–2017) conducted in a tertiary pediatric hospital in Singapore reported 13 cases of patients with  samples . A 15.4%E. meningoseptica infection in a retrospective study conducted between January 2011 and June 2012 at the Singapore General Hospital (SGH). The patient was among 118 adult hemodialysis patients confirmed with vascular-access-associated bloodstream infection (VAABSI) [A hemodialysis patient was confirmed positive for (VAABSI) .E. meningoseptica infection. Antibiotic therapy was changed to rifampicin and piperacillin/tazobactam for a period of 4 weeks until full recovery [Another case involved a preterm infant born with marked generalized abdominal distension and respiratory distress in the third trimester (33rd week), who was transferred to the neonatal intensive care unit for further care, and three weeks later, the patient was diagnosed with sepsis and subsequently, cloxacillin and amikacin were initiated. Blood samples were collected, and the patient was confirmed to be positive for recovery .E. meningoseptica patients in Singapore. The most commonly used combination is piperacillin/tazobactam with trimethoprim/sulfamethoxazole, followed by piperacillin/tazobactam with fluroquinolone [Combined antibiotic therapy is reported to be the choice of treatment for uinolone ,95. Otheuinolone ,23, clinuinolone .cps cluster [mutY gene caused by the insertion of an integrative and conjugative element (ICEEa1). Genetic and morphological changes could have substantially contributed to the evolutionary dynamics of the outbreak strains that could have increased their concomitant adaptability eventuating in a hypermutator phenotype [Singapore and Wisconsin outbreak isolates have type I  cluster . Additiohenotype . These “henotype .Elizabethkingia infections from Thailand. Nevertheless, a retrospective study conducted by researchers from the University of Bangkok and Chulalongkorn University among eight hospitalized patients at King Chulalongkorn Memorial Hospital (KCMH) led to the first reported cases of E. meningoseptica infection in Thailand [E. meningoseptica [In comparison with Malaysia and Singapore, there are not as many reports on Thailand . All isoThailand . A subseoseptica .Elizabethkingia spp. infections in Indonesia only appeared in the past couple of years. In Malang, Indonesia, a three-month-old infant presented to the Emergency Department of a tertiary hospital with a history of a 15-day fever associated with lethargy [E. meningoseptica using the VITEK 2 system (bioMérieux). The isolate was further tested with various antimicrobials and showed susceptibility to cefepime, tigecycline, trimethoprim-sulfamethoxazole, intermediate susceptibility to combined antibiotics piperacillin-tazobactam, and resistance to ampicillin, ampicillin-sulbactam, cefazolin, ceftazidime, aztreonam, meropenem, amikacin, and gentamicin. The patient was put on three weeks cefepime (50 mg/kg per body weight/day) followed by mechanical ventilation, fluid and electrolyte therapy, intravenous hypertonic saline infusion, intravenous inotropic therapy, thrombocyte concentrate and fresh frozen plasma transfusion, and oral fluid cortisone therapy. After eight weeks of treatment, the patient showed clinical improvement.Reports of lethargy . The patE. meningoseptica in one non-dengue febrile patient [E. meningoseptica from one of their patients’ blood samples diagnosed with septicemia [E. meningoseptica from their blood samples, although the method of identification was not stated [In Jakarta, a group of researchers from the Eijkman Institute reported the presence of  patient . A retropticemia . In a crt stated .Elizabethkingia spp. infections in Cambodia are rare. Reed et al. (2020) reported a case in which a 7-day-old female patient with presumed late-onset neonatal sepsis was transferred to the pediatric ICU at Angkor Hospital for Children, Siem Reap, Cambodia [E. anophelis through MALDI-TOF MS using the bioMérieux VITEK MS. Later, the patient’s antibiotic therapy was replaced with intravenous ciprofloxacin  and vancomycin , and the patient was discharged after 28 days. A month later, the patient was hospitalized, and clinical features showed raised intracranial pressure, including neurologic deficits. The patient was later diagnosed with hydrocephalus. This had led the researchers to the retrieval of the seven isolates stored at −80 °C from January 2012 to October 2018 that had been previously identified as C. meningosepticum, C. miricola, or Elizabethkingia spp. Isolates were subcultured and analyzed using VITEK MS MALDI-TOF mass spectrometry; six of them were re-identified as E. anophelis and one isolate as E. meningoseptica. It is worth noting that four out of seven patients had died due to ventilator-associated pneumonia (VAP) and sepsis [Reports of Cambodia . The patd sepsis .Elizabethkingia spp. infections in Southeast Asia showed the dearth of knowledge in the majority of countries within the region. Of the eleven Southeast Asian nations, only Singapore had the most data and published reports, while we were unable to find any clinical case reports from the Philippines, Brunei, Vietnam, Laos, Myanmar, and Timor-Leste. Genomic data are also available mainly from Singapore, with currently only a single isolate of E. anophelis from Malaysia in GenBank (accession no. JNCG01000000) that was (erroneously) deposited in 2014 as E. meningoseptica. The paucity of our knowledge is an important challenge when dealing with Elizabethkingia infections and requires urgent attention from researchers and medical and health officials. Elizabethkingia spp. is still an understudied pathogen with an intrinsic multidrug resistance phenotype that has been reported in several countries around the world, causing opportunistic infections with high mortality rates. The apparent ubiquity of the pathogen, being found in diverse environments and animal hosts, and the presence of multiple antimicrobial resistance genes in its genome is a cause of serious concern as it could serve as a natural reservoir of antimicrobial resistance genes for horizontal transmission to other pathogenic microorganisms. Since currently, only whole-genome sequencing  is able to accurately identify Elizabethkingia to the species level, cheaper but equally specific and sensitive identification or diagnostic methods need to be developed, as genome sequencing and MALDI-TOF MS are not available to most hospital diagnostic laboratories in poorer Southeast Asian countries. Nevertheless, a deeper understanding of the pathogen along with more precise diagnostic procedures, better accessibility of treatment options either with antibiotics or other alternatives such as bacteriophage therapy, and improvements in the prevention of transmission and infection will eventually enable better control of this extremely pathogenic and highly resistant bacteria.Our review of case reports involving