Aeromonas species exhibit widespread presence in food, poultry, and aquaculture. They are major multi-drug-resistant fish pathogens. This study aims to identify Aeromonas species harbouring virulence genes aerolysin, flagellin, and lipase from diseased fishes of Assam wetlands with association with antibiotic resistance and in vivo pathogenicity. One hundred and thirty-four Aeromonas strains were isolated and thirty representative species identified using genus-specific 16S rRNA gene amplification. A. veronii was most prevalent (53.7%) followed by A. hydrophila (40.2%), A. caviae (4.47%), and A. dhakensis (1.49%). Ninety percent (90%) of strains harboured at least one of the studied virulence genes: aerA (73.3%), lip (46.6%), and flaA (26.6%). The highest multiple antibiotic resistance (MAR) index 0.8 corresponded to A. hydrophila DBTNE1 (MZ723069), containing all the studied genes. The lowest LD50 values (1.6 × 106 CFU/fish) corresponded to isolates having both aerA and lip. β-lactams showed utmost resistance and lowest for aminoglycosides. There was a significant (p < 0.05) Pearson chi-square test of association between the occurrence of virulence and antibiotic resistance. The in silico protein–protein interaction revealed important drug targets, such as σ28 transcription factor, aminoacyl-tRNA synthetase, and diacylglycerol kinase, with significant (p < 0.05) enrichment. This study suggests that fish-isolate Aeromonas strains represent potential threat to aquaculture with subsequent risk of transferring antibiotic resistance to human pathogens. Amblypharyngodon mola, Puntius spp., Bangana dero, Colisa spp., Chanda spp., Mystus vittatus, Ambassis spp., Mastacembelus armatus, Labeo rohita, L. catla, L. bata, Ompok bimaculatus, Channa punctatus, Clarias batrachus, Wallgo attu, small prawns, etc.  = 18.687 for MAR and aerA, X2(10) = 23.112 for MAR and lip, and X2(10) = 30.0 for MAR and flaA. Goodman and strength of association between variables were strong with a Cramer’s V value of >0.4. Scatter plot of strains (n = 30) vs. MAR value showed presence and absence of genes  for the isolates containing both the aerolysin and lipase genes was lower with an average of 1.6 × 106 CFU/fish. The highest dose corresponded to isolates containing flagellin gene with an average LD50 1.42 × 107 CFU/fish. Among the isolates, DBTNE9 (MZ723498) had the lowest LD50 1.05 × 106 CFU/fish. Isolates expressing the lipase protein was found to be more virulent than aerolysin and flagellin , prolyl tRNA synthetases with a Stringdb score of 0.719. The second Stringdb score belonged to the interaction with peptidase M4 of the thermolysin family (0.712). The PPI enrichment was confirmed to be significant . AveragefliD flagellar hook-associated protein (Stringdb score: 0.975) yielded strongest interaction with flagellin. RNA polymerase sigma factor: fliA scored the second-best interaction (0.963). All 11 genes in the network were co-occurring and co-expressing. The PPI enrichment was significant with a clustering coefficient value of 1  with its co-expressing gene, chaperon protein (Lip_chaperone). The second best interaction (0.907) was found with diacylglycerol kinase (dgkA) (0.907). Two co-occurring genes and two co-expressing genes were described along with of 0.895 . The degof 0.895 .Aeromonas spp. is a result of triad interactions between the bacteria, fish, and aquatic environment. To better understand the pathogenesis details of the disease caused by Aeromonas spp. and their resistance to commonly used antibiotics, a comparative analysis of the virulence factors and the antibiogram of the bacteria were studied together. In the present study, the highest number was recorded in A. veronii (53.7%), followed by A. hydrophila (40.2%). The result is similar with the findings of Sun et al. (2016) [Aeromonas isolates from other habitats and contaminated food were mostly from A. caviae, followed by A. dhakensis [A. veronii, A. hydrophila, and A. caviae are the major aeromonads causing MAS in the freshwater aquaculture system of Assam, India [A. veronii and A. hydrophila strains display high potential of pathogenicity in fish [Aeromonas strains were carrying significant virulence genes. However, 6.6% of the Aeromonas strains mostly isolated from S. sarana, carried none of the tested virulence genes. However, there are scanty reports of an Aeromonas infection in S. sarana. This could potentially be the reason why the bacteria failed to cause extreme septicemic manifestations in the particular fish host  , Foysal . (2016) , and Li . (2016) . In conthakensis . A. verom, India . Pathogem, India . Result  in fish . Ninety ish host A. Hence,aerA gene, which was most prevalent among all the 30 species with the highest occurrence in A. veronii (52.17%). Gene codes for aerolysin, a pore-forming toxin causing hemolysis and soft tissue necrosis in the intestine of host [Aeromonas spp. has been also reported earlier [lip) is another virulence factor present in a broad range of Aeromonas spp. Gene alters the membrane permeability of the host tissues and facilitates bacterial colonization and proliferation [lip gene, of which 42.85% were A. hydrophila strains from tilapia and carp. There are earlier studies on the prevalence of lip gene in 23.1% of A. hydrophila isolates of Nile tilapia [flaA gene encoding flagellin was harboured by 26.6% of the strains, of which 10% of each was A. veronii and A. hydrophila, and 6.6% constituted of A. caviae. Polar flagellum of the Aeromonas strains consists of two tandem subunits flaA and flaB, which polymerize to form the filaments [In the present investigation, 76.6% of the strains carried the  veronii .17%. Gen earlier ,54. Extrferation . In this tilapia . Flagell tilapia . In the ilaments .aerA, lip, and flaA with their encoded proteins interact with several other proteins involved in the signaling pathways leading to pathogenesis [A. veronii interacted strongly with DBP prolyl-tRNA synthetase (0.71) and its smaller paralog, YbaK. Prolyl-tRNA synthetase belongs to the ubiquitously expressed enzyme family of aminoacyl-tRNA synthetases (aaRS), performing a crucial role in protein biosynthesis, cell growth, and viability. Enzyme of bacterial origin is different from the eukaryotic one and widely studied as a promising antimicrobial drug target [Vibrio cholera, Enterococcus faecalis, Pseudomonas aeruginosa, and Clostridium perfringens. Enzyme also provides stability to microorganisms to thrive under extremely adverse growth conditions. High-affinity inhibitors of the M4 protease family opened new therapeutic ways in clinical practice and computational biochemistry [A.hydrophila from tilapia [Tested virulence genes ogenesis . In thisg target . Second g target . Peptidag target . It is ahemistry ,63. Nonahemistry . In our  tilapia .Aeromonas strains interacts with 16 genes based in previous studies [flgE, flgK, fliS, and fliD), rod , biosynthesis (flhA), motor switch (fliG), and σ28factor (fliA). FliD protein with the strongest interaction (0.97) forms the basal part of the flagella regulating the mucin-specific adhesion of the bacteria on the host surface [fliA) is an integral part of the flagellin gene network encoding the Aeromonas spp. polar flagellum. The fliA mutation leads to polar flagellar dysfunction and biofilm formation failure by Aeromonas spp. The fliA gene is responsible for regulating flagellin adherence and biofilm formation potential.Lipase shows its highest interaction with lipase chaperon (0.929) that helps in its proper folding and release through the periplasm in its active protease-resistant form . Kinase  studies , of whic surface . The σ28Aeromonas [Aeromonas spp. to beta-lactam antibiotics [A. veronii and 22.1% A. hydrophila) were showing the highest resistance to third generation cephalosporins (cefixime (56.6%) and cefotaxime (53.3%)) followed by fourth generation cefepime (46.6%). This phenomenon may be attributed to the presence of Class C cephalosporinases [Escherichia coli isolated from German ICUs [A. hydrophila strains exhibited low resistance against the combination of cefepime and clavulanic acid (CFC) due to the synergistic effect of both the antibiotics. Matter of fact, 82.35% of the cephalosporin-resistant strains contained at least one of the tested virulence genes. Our studies showed that all Aeromonas strains were susceptible to aminoglycosides except for A. dhakensis strain 21 and A. veronii strain 22 containing all the three virulence genes. These observations predict the correlation of antimicrobial resistance and presence of virulence factors. Earlier studies also reported the susceptibility of Aeromonas strains cultured in vitro and those obtained from natural water bodies to aminoglycoside antibiotics [Aeromonas strains were sensitive to quinolones and fluoroquinolones except for A. caviae. This phenomenon could be explained based on Type II topoisomerase quinolone resistance-determining regions of A. caviae. All the tested Aeromonas strains exhibited multidrug resistance with a MAR index of >0.2, which might be due to the presence of different virulence genes, antibiotic resistance genes, or exposure to antimicrobials in the environment [A. veronii strain 6, which also carried all the tested virulence genes. In a recent study, 36% isolates of Aeromonas strains isolated from diseased rainbow trout were found to be multi-antibiotic resistant [Aeromonas spp. showed increased resistance to carbapenem, aztreonam, and third generation and fourth generation cephalosporins [The pathogenicity of the isolates was found to be a direct measurement to virulence and toxicity. The co-existence of aerolysin and lipase was observed to be more lethal to fish. The presence of flagellin was comparatively avirulent. The study is in agreement with a previous study that used a mouse model challenged orally with eromonas . Antibioibiotics . Most storinases . These sman ICUs . Moreoveibiotics . This meibiotics  while thibiotics . All theironment . MAR indesistant . In anotosporins .Aeromonas spp. to adapt to the changing environmental conditions. The aerolysin gene is the most frequently distributed among the tested Aeromonas strains followed by flagellin and lipase gene. All the strains with at least one virulence gene were multidrug-resistant. This suggests a positive correlation between the presence of the virulence genes and antibiotic resistance profile in the Aeromonas strains of diseased fish origin. Cephalosporins (fourth generation) and aminoglycosides were comparatively effective against the Aeromonas strains. The cefepime and clavulanic acid combination (CFC) also showed inhibitory results against the tested strains. The six strains  isolated from aquaculture farms were mainly A. veronii, which had comparatively less resistance profile than wetland strains. The probability of using potential antibiotics in studied wetlands is very less. Therefore, the possibility of antibiotic leaching from other sources as inappropriate disposal of medical wastes cannot be ruled out. The findings of this study are important for the surveillance of pathogenic-resistant Aeromonas strains, which could potentially threaten human and animal health. This study also shows that there is an antibiotic resistance pattern in the wetland systems which needs to be further investigated.The divergence in the virulence profiles and antibiotic susceptibility patterns in different water bodies of Assam highlights the ability of


Pinellia ternata is an important traditional medicine in China, and its growth is regulated by the transcriptome or proteome. Lysine crotonylation, a newly identified and important type of posttranslational modification, plays a key role in many aspects of cell metabolism. However, little is known about its functions in Pinellia ternata.Pinellia ternata and examined its overlap with lysine succinylation. A total of 2106 crotonylated sites matched on 1006 proteins overlapping in three independent tests were identified, and we found three specific amino acids surrounding crotonylation sites in Pinellia ternata: KcrF, K***Y**Kcr and Kcr****R. Gene Ontology (GO) and KEGG pathway enrichment analyses showed that two crucial alkaloid biosynthesis-related enzymes and many stress-related proteins were also highly crotonylated. Furthermore, several enzymes participating in carbohydrate metabolism pathways were found to exhibit both lysine crotonylation and succinylation modifications.In this study, we generated a global crotonylome analysis of Pinellia ternata, especially in the biosynthesis of alkaloids, and some metabolic pathways are simultaneously regulated by lysine crotonylation and succinylation.These results indicate that lysine crotonylation performs important functions in many biological processes in The online version contains supplementary material available at 10.1186/s12870-022-03835-y. Posttranslational modifications (PTMs), e.g. the introduction of novel functional groups on lysine residues, such as acetyl, phosphoryl, ubiquityl, succinyl, crotonyl and methyl groups , often oLysine crotonylation (Kcr) was first identified on histone proteins and mainly occurs at the ε-amino group of lysine, which is a chemical modification similar to acetylation , 3. PrevPinellia ternata (Thunb.) Beri is a perennial herb belonging to Araceae. It has been widely utilized as an important component of traditional Chinese medicine for thousands of years. The tuber of P. ternata exhibits specific pharmacological properties, such as antiemetic, expectorant, antipyretic and styptic effects [P. ternata is increasing in China, sources are becoming scarce because of overexploitation. With the development of bioinformatics, molecular biology, and proteomics, large amounts of omics analysis have been performed on P. ternata. The transcriptome of P. ternata under normal and shaded environments provided a foundation for further study of this major traditional herb [ effects –17. Althnal herb . Zhu et nal herb .P. ternata has been reported. In this study, we performed the first proteome-wide analysis of lysine crotonylation in the leaves of P. ternata with the purpose of providing more information about metabolic regulation in the biosynthesis of ephedrine alkaloids. This study may offer insights into the functional characterization of lysine crotonylation in P. ternata.To date, no global analysis of lysine crotonylation in P. ternata. To investigate the changes in lysine crotonylation sites, the proteins of P. ternata leaves were extracted and digested into peptides by trypsin. Consistent with the properties of tryptic peptides, the length of most peptides ranged from 7 to 20 amino acids   Fig. a. All th tobacco –28. It icr sites, a heatmap was generated. In accordance with the conserved motifs KcrF and FKcr, phenylalanine (F) was enriched in the + 1 and -1 positions near crotonylation sites, suggesting that it is a widespread amino acid around the crotonylated sites  functional classification analysis was performed  and cytoplasm 35.42%). Meanwhile, some proteins were predicted to be distributed in the nucleus (11.24%), mitochondria (6.97%), and plasma membrane (2.79%)  and acetolactate synthase (AHAS) had one and two crotonylation sites, respectively , are necessary for plant development . In our P. ternata, and 356 sites in 161 proteins were found to be succinylated. To elucidate the relationship between crotonylation and succinylation of the same lysine residue, the lysine crotonylome and the succinylome were both analysed. A total of 128 proteins and 206 sites were modified by both crotonylation and succinylation  and five subunits of photosystem II , were found to be crotonylated  to form glucose, which also acts as the product of photosynthesis. There are many key enzymes involved in carbon fixation, such as ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), fructose-1,6-bisphosphatase (FBPase) and phosphoenolpyruvate carboxylase (PEPC) –31. PrevP. ternata , which are crucial chaperonins , 39. HSPP. ternata, among which ephedrine is the main ingredient that belongs to phenylpropylamino alkaloids. Studies have shown that ephedrine plays an important role in clinical treatment, such as bronchial asthma, nasal mucosal congestion and nasal congestion [P. ternata by increasing the content of ephedrine.Alkaloids are one of the most important medicinal compounds in ngestion , 42. TheP. ternata requires further investigation.The biosynthesis of ephedrine comprises complex branching biochemical pathways , gibberellic acid (GA), ethylene, cytokinin (CTK), and brassinosteroids (BRs), play vital roles in the regulation of plant growth and development . Many stP. ternata remains to be elucidated.ABA, another important phytohormone, functions in many aspects of plant development, such as seed germination, dormancy, seeding growth and seed maturation . A numbeP. ternata. Our data showed that the proteins related to the oxidative phosphorylation pathway, pentose phosphate pathway, glycolysis/gluconeogenesis pathway, TCA cycle pathway, glycine, serine and threonine metabolism pathway, glyoxylate and dicarboxylate metabolism pathway, carbon fixation in photosynthetic organisms pathway, and photosynthesis pathway displayed an increased tendency to be both crotonylated and succinylated in the KEGG pathway analysis  used in this study were collected from Gansu, China (34.0°N latitude and 105.3°E longitude). The plant specimen was deposited in the Chinese Virtual Herbarium with an herbarium ID of WUK 5,971,434 (http://ppbc.iplant.cn/), and the tubers were identified by Renbin Zhu. The tubers were planted in nutrient soil in a greenhouse with a relative humidity of 50% and a temperature of 22 °C during the day and 18 °C at night. The leaves of three-week-old seedlings were collected, snap-frozen in liquid nitrogen and stored at -80 °C for crotonylome analysis.Thetubers of Pinellia ternata leaves were extracted as described in previous studies [Proteins from  studies , 54, 55. studies . The sup studies . After aAfter trypsin digestion, the peptides were fractionated into 80 fractions by high pH reverse-phase HPLC using Ultimate RSLCnano 3000. For affinity enrichment, the fractions of peptide were incubated with pan anti-crotony lysine antibody beads . The lysThe enriched crotonylated peptides were investigated by mass spectrometry as described previously , 54, 55.P. ternata (sequenced by Micrometer Biotech Company). The parameters in MaxQuant were set according to Guo et al. (2020) [The MS/MS data of crotonylated peptides were processed through MaxQuant with the integrated Andromeda search engine (v.1.5.2.8) . The tan. (2020) .http://www.ebi.ac.uk/GOA/). The domains of proteins were identified by InterProScan  based on the protein sequence alignment method, and the InterPro domain database was used. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and WoLF PSORT databases were used to annotate protein pathways and subcellular localization respectively. Motif analysis of lysine acetylation sites was performed by MoMo  software. Enrichment-based clustering analysis was performed with the R-package following a previous study [The Gene Ontology (GO) annotation proteome was derived from the UniProt-GOA database , Distribution of Kcr-modified proteins based on the number of crotonylation sites in a protein (b). The analysis was performed based on 2106 crotonylated sites matched on 1006 proteins overlapping in three independent tests.Additional file 2: Supplementary Table S1. Crotonylated sites of proteins in the leaves of P. ternata.Additional file 3: Supplementary Table S2. Functional classification of crotonylated proteins in the leaves of P. ternata. Additional file 4: Supplementary Table S3. GO enrichment analysis of crotonylated proteins in the leaves of P. ternata. Additional file 5: Supplementary Table S4. KEGG pathway analysis of crotonylated proteins in the leaves of P. ternata. Additional file 6: Supplementary Table S5. Protein domain enrichment analysis of crotonylated proteins in the leaves of P. ternata. Additional file 7: Supplementary Table S6. Annotation of the total proteins in the leaves of P. ternata.


Clostridioides difficile infections in Korea, there has been an increase in inappropriate testing for C. difficile, which has rendered overdiagnosis of asymptomatic colonisers common. We aimed to investigate the appropriateness of C. difficile testing and the related factors.Given the increasing incidence of C. difficile infection from September 2019 to November 2019. We performed logistic regression analysis to investigate factors related to inappropriate testing. Further, a survey was conducted on physicians to assess the knowledge and ordering patterns of C. difficile testing.We retrospectively reviewed the medical records of patients who were admitted to a 1300-bed tertiary-care teaching hospital in Korea and were tested for C. difficile tests were conducted in the emergency room  but decreased in intensive care units . The survey was conducted on 61 physicians. Internal medicine physicians had significantly higher scores than non-internal medicine physicians . The most frequently ordered combination of tests was toxin + glutamate dehydrogenase (47.5%), which was consistent with the ordered tests.We included 715 tests from 520 patients in the analysis. Testing was classified as hospital-onset and community-onset and subclassified as appropriate and inappropriate following an algorithmic method. Among the 715 tests, 576 (80.6%) and 139 (19.6%) tests were classified as hospital-onset and community-onset, respectively. Among the hospital-onset tests, 297 (52%) were considered inappropriate. The risk of inappropriate testing increased when C. difficile tests were performed inappropriately. The patient being located in the emergency room and intensive care unit increased and decreased the risk of inappropriate testing, respectively. In a questionnaire survey, we showed that internal medicine physicians were more knowledgeable about C. difficile testing than non-internal medicine physicians. There is a need to implement the diagnostic stewardship for C. difficile, especially through educational interventions for emergency room and non-internal medicine physicians.Almost half of the The online version contains supplementary material available at 10.1186/s13756-022-01111-0. Clostridioides difficile infection (CDI) is among the most common cause of healthcare-associated infections [fections . The prefections . Recent fections ; howeverfections .C. difficile shows various clinical presentations, which range from asymptomatic colonisation to a life-threatening infection [C. difficile is 3% to 26% and 5% to 7% in acute care hospitals [C. difficile nucleic acid amplification test (NAAT) and glutamate dehydrogenase (GDH), the currently used methods cannot discriminate between CDI and asymptomatic colonisation with C. difficile [nfection , 6. The ospitals  and longospitals , 11, resifficile , which rifficile –15. Therifficile .C. difficile testing in a Korean teaching hospital. Further, we aimed to evaluate risk factors associated with inappropriate testing. Finally, we aimed to conduct a questionnaire survey of physicians to assess their knowledge and actual ordering patterns in order to investigate physician-related risk factors for inappropriate C. difficile testing.In 2017, the Infectious Diseases Society of America (IDSA) and the Society for Healthcare Epidemiology of America updated the clinical practice guidelines for CDI and recommended testing in the relevant clinical settings (unexplained and new-onset ≥ 3 unformed stools within 24 h) for improved reliability of laboratory tests. Additionally, they suggested appropriate diagnostic methods . This stC. difficile testing. C. difficile toxin enzyme immunoassay (EIA), C. difficile GDH EIA, C. difficile NAAT, and C. difficile culture were included as C. difficile tests. C. difficile toxin EIA was performed using VIDAS C. difficile A & B  while C. difficile GDH EIA was performed using VIDAS C. difficile GDH . C. difficile NAAT was conducted using the BD Max Cdiff assay . C. difficile culture was performed using ChromID C. difficile agar . The exclusion criteria were as follows: age < 18 years, C. difficile tests performed in outpatient settings, or having undergone colostomy.We retrospectively reviewed the electronic medical records of patients who were admitted to a 1300-bed tertiary-care teaching hospital in Korea between 1 September 2019 and 30 November 2019. Eligible patients included those who underwent C. difficile infection [Testing for hospital-onset CDI and community-onset CDI was separately analysed. Testing for hospital-onset CDI was defined as tests performed > 3 days after admission or transfer from other hospital; testing for community-onset CDI was defined as tests performed ≤ 3 days after admission . The appnfection . Tests wShock was defined as mean arterial pressure < 65 mmHg or using a vasopressor on the testing day. Ileus was defined through simple abdominal radiography performed on the testing day and confirmed by a radiologist.C. difficile testing were classified as internal medicine and non-internal medicine physicians since almost half of the C. difficile tests were ordered by internal medicine physicians. Further, we assumed that internal medicine physicians and non-internal medicine physicians showed different clinical practices regarding CDI-associated symptoms.Comorbidity was determined using the Charlson comorbidity index . PatientWhite blood cell count of 15,000 cells/mL and serum creatinine level of 1.5 mg/dL were used to indicate severe infection according to the severe CDI criteria in IDSA guidelines . Fever wC. difficile testing between September 2019 and November 2019 to assess their knowledge and ordering patterns. The survey included ten questions evaluating knowledge and one question assessing the test ordering patterns , with comparisons using Student’s t-test. For further analysis of between-speciality differences in the scores, the scores were categorised into quartiles; further, the proportion of participants with the highest quartile was compared. The correct rate of each question was expressed as a percentage, with between-speciality comparisons being performed using the chi-square test. Statistical analyses were performed using IBM SPSS Statistics for Windows, ver. 22.0 . Statistical significance was set at P < 0.05.C. difficile tests were performed in 691 patients. Among them, we excluded patients aged < 18 years (n = 177), patients who underwent C. difficile testing in an outpatient setting (n = 72), and patients who underwent colostomy (n = 6). Finally, 715 tests from 520 patients were included . In the appropriate testing group, 243 (81.8%), 53 (17.8%), and 1 (0.3%) tests were conducted in the general ward, intensive care unit, and emergency room, respectively, while the corresponding values in the inappropriate testing group were 242 (86.7%), 23 (8.2%), and 14 (5.0%) . Compared with the inappropriate testing group, the appropriate testing group had a significantly higher proportion of patients with unformed stool ≥ 3 times within 24 h , shock , and ileus , as well as a lower proportion of patients who used laxative within 48 h . The appropriate testing group had a significantly higher proportion of internal medicine physicians than that in the inappropriate testing group . There were no between-group differences in the proportion of patients with indicators for severe infection, albumin < 3 g/dl, fever, history of gastrointestinal surgery within 30 days, history of antibiotic use over the past 30 days, transfer from other hospital. There was no difference between the two groups in the median number of days between admission and testing.In the hospital-onset setting, 297 51.6%) and 279 (48.4%) tests were classified as appropriate and inappropriate, respectively. Table .6% and 2C. difficile tests conducted in the emergency room  and decreased risk in the intensive care unit . Tests ordered by non-internal medicine physicians were not significantly associated with inappropriate testing . Sex, age, and the Charlson comorbidity score were not associated with the appropriateness of C. difficile testing.We performed multivariate logistic regression analysis of hospital-onset CDI with the inclusion of sex, age, and the characteristics with significant between-group differences  Table . There wIn the community-onset setting, 65 (46.8%) tests were appropriate and 72 tests (53.2%) were inappropriate. No risk factor was found in the multivariate logistic analysis , general surgery , emergency medicine , neurosurgery , neurology , and others . The mean score for the total population was 6.4 out of 10. Internal medicine physicians showed significantly higher scores than non-internal medicine physicians   physicians reported ordering Toxin EIA + GDH EIA as the initial C. difficile test ; moreover, 274 (38.3%) tests included C. difficile culture.Regarding the ordering pattern of st Table , while nC. difficile testing than non-internal medicine physicians in the questionnaire. The most frequently ordered tests were Toxin EIA and GDH EIA.Our findings demonstrated that almost half of the tests were performed inappropriately. Inappropriate testing was significantly associated with patient location. Tests performed in the emergency room increased the risk of inappropriate testing, while those performed in the intensive care unit decreased the risk. Internal medicine physicians had a significantly higher proportion of appropriate tests in univariate analysis; however, physician specialty was not statistically significant risk factor of inappropriate testing in multivariate analysis. Nevertheless, internal medicine physicians were more knowledgeable about C. difficile testing [There have been inconsistent reports regarding the appropriateness of  testing , 18. Kel testing  and Kara testing . HoweverC. difficile testing, especially in emergency room and non-internal medicine physicians, could reduce unnecessary testing and treatment.Our findings demonstrated that the location where the tests were conducted influenced the test appropriateness. Performing tests in the emergency room increased the risk of inappropriate testing. In addition to patient factors, including altered mentality, patient handoffs commonly occur in the emergency room , which iC. difficile testing in community-onset setting partly because of the small number of cases. Since the importance of community-onset CDI is increasing, additional large-scale studies are needed.Our study showed that about 20% of CDI testing was conducted in community-onset settings. When judging by the history of antibiotics use, laxative use, shock, ileus, and, the number of diarrhoea, the proportion of appropriate CDI testing in community-onset was 46.8%. In Korea, 12.1% of CDI were reported to be community-onset CDI . SimilarC. difficile testing by comparing the survey answers and actual orders. Approximately 50% of the physicians responded that they ordered toxin EIA + GDH EIA as the initial test for CDI diagnosis, which was comparable with the actual orders. However, NAAT was rarely used in primary testing possibly because it could be the second step of a two-step approach. IDSA and European guidelines recommend a multi-step algorithm  [C. difficile and only colonization, although it may facilitate the analysis of epidemiological data, such as the proportion of C. difficile colonizers. Our findings suggest the need for diagnostic stewardship for appropriate clinical situations and proper testing methods.We examined the ordering pattern of by NAAT) , 27. In by NAAT) . InsteadC. difficile testing in Korea. Further, we analysed data from a relatively large number of patients in this field. Additionally, analysis of both patient- and physician-related risk factors showed that more targeted diagnostic stewardship for emergency rooms or non-internal medicine physicians could effectively reduce unnecessary testing and treatment.Our study has several limitations. First, this was a retrospective study; therefore, the medical records may be inaccurate due to information bias. Second, this was a short-term, single-centre, non-interventional observational study; therefore, our findings may not be generalisable to other hospitals. Despite these limitations, our study has several strengths. To our knowledge, this is the first study to investigate the appropriateness and physician practice patterns of C. difficile tests were performed inappropriately. Diagnostic stewardship for CDI should be implemented, particularly through educational interventions for those working in the emergency room and for non-internal medicine physicians. Prospective multicentre studies on the effectiveness of diagnostic stewardship are warranted.Our findings showed that almost half of the Additional file 1. Method S1. The questionnaire for diagnosis of Clostridioides difficile infection (CDI). Table S1. Multivariate logistic regression analyses for inappropriate Clostridioides difficile testing in community-onset settings. Fig. S1. Percentage of corrected answers to questions about knowledge of CDI testing.


Tripaphylus Richiardi in Anonymous, 1878 (family Sphyriidae) are described from elasmobranch hosts caught as bycatch within the Demersal and Timor Reef Fisheries which operate in the Northern Territory exclusive economic zone. Tripaphylus squidwardin. sp. was collected from Carcharhinus coatesi Whitley and had a prevalence of 11.6%. Tripaphylus dippenaaraen. sp. was collected from Rhizoprionodon acutus (Rüppell) and had a prevalence of 28.2%. The new species are distinguished from existing congeneric species by the body proportions and shape of the adult female and by the arrangement of lobes on the ventral surface of the cephalothorax.Two new species of the genus  Sphyrion laevigatum  was the first to be reported from Australian waters. It was recorded under the name Sphyrion australicus Thor, 1900, and was considered to be a new species by Thor .The copepod family Sphyriidae comprises 42 valid species classified in eight genera  was originally described from Glaucostegus typus (Anonymous [Bennett])  in southeastern Queensland  was described from two species of Asymbolus, A. analis (Ogilby) and A. rubiginosus Last, Gomon & Gledhill, caught off southeastern Queensland  collected from the ventral throat region of the host. These specimens constitute the type series. A further 20 non-type specimens of Tripaphylus were collected as part of the project. The total of 46 Tripaphylus specimens came from 27 infected hosts  with a mean total length of 707.8 mm: the prevalence was 11.6%, the mean intensity was 1.7 (ranging from 1 to 6 per host).The description of the new Tripaphylus species from Rhizoprionodon acutus (Rüppell) is based on the examination of 4 specimens , collected from the ventral throat region of the host. These specimens constitute the type series. A further 7 non-type specimens of Tripaphylus were collected as part of the project. The total of 11 Tripaphylus specimens came from 11 infected hosts  with a mean total length of 755.8 mm: the prevalence was 28.2%, the mean intensity was 1.0.This description of the new Specimens were cleared in lactic acid and observed whole on a Leitz dissecting microscope. Dissected appendages were examined on an Olympus BH2 microscope using differential interference contrast. Clotted host blood was present on the appendages of most specimens obscuring detail. Drawings were made using a drawing tube and measurements were made using a stage micrometer. Morphological terminology conforms to Kabata  and HuysC. coatesi and one from R. acutus were examined using scanning electron microscopy (SEM). They were washed and dehydrated overnight in a series of graded ethanol solutions , paratypes are deposited in MAGNT and in the Natural History Museum, London (NHM),Family SphyriidaeTripaphylus Richiardi in Anonymous, 1878Genus Tripaphylus squidwardi new speciesType Material.Carcharhinus coatesi Whitley, 1939 , MAGNT Registration Number NTM Cr019479; paratype intact female and 2 headless paratype females from same host (CARCOA 400), NTM Cr0179480; paratype female intact but lacking egg sacs, from C. coatesi , NTM Cr0179481; paratype female intact but lacking egg sacs, from C. coatesi , NTM Cr0179482; 2 paratype females intact and bearing egg sacs, from C. coatesi , NTM Cr0179483; 4 paratype females intact, 3 bearing egg sacs, and 3 incomplete paratype females from C. coatesi , NTM Cr0179484; paratype female intact with egg sacs, from C. coatesi , NTM Cr0179485; paratype female intact with egg sacs, from C. coatesi , NTM Cr0179486; headless paratype female, trunk bearing paired egg sacs, from C. coatesi , NTM Cr0179487; headless paratype female, trunk lacking egg sacs, from C. coatesi , NTM Cr0179488; 3 headless paratype females, 1 ovigerous and 2 with trunk lacking egg sacs, from C. coatesi , NTM Cr0179489; headless paratype female, trunk with broken egg sacs, from C. coatesi , NTM Cr0179490; headless paratype female, trunk with broken egg sacs, from C. coatesi , NTM Cr0179491; 2 isolated paratype heads plus 3 headless trunks lacking egg sacs, from C. coatesi , NTM Cr0179492; headless paratype female, trunk lacking egg sacs, from C. coatesi , NTM Cr0179493; headless paratype female, trunk lacking egg sacs, from C. coatesi , NTM Cr0179494; headless paratype female, trunk lacking egg sacs, from C. coatesi , NTM Cr0179495; paratype female with head intact but trunk incomplete, from C. coatesi , NTM Cr0179496: 4 paratype females intact, 3 bearing egg sacs, and 3 incomplete paratype females from C. coatesi  NHMUK 2022.174-177.Holotype female, intact with one missing egg sac, from LSID urn:lsid:zoobank.org:act:4836F799-E8B6-4C46-A2CB-8E31F8FF854DTransformed adult female. Body Fig. A comprisNeck slender, cylindrical with smooth margin; expanded anteriorly at junction with cephalothorax; expanding gradually posteriorly to merge with trunk. Neck always longer than trunk, mean 1.48 times, with a range of 1.02 to 1.90 times longer (n = 11). Trunk about 4.3 times longer than wide, slightly compressed dorsoventrally and increasing in width towards maximum at posterior end; bearing paired lateral swellings near posterior end , have an unequivocal demarcation between the neck and the trunk  and T. beatricae Dippenaar, 2018, the trunk is reasonably clearly defined and is short  , as in T. beatricae, the posterior processes are distinctly longer than the trunk whereas in the new species these processes are shorter than the trunk. Distinguishing the new species from the remaining six valid species relies primarily on the number and configuration of the lobes on the cephalothorax, as well as the length of the posterior processes relative to the trunk.The general body shape and body proportions of the transformed adult female of the new species are similar to several other congeneric species. Only two species,  Kabata, , marked Tripaphylus vassierei  has a complex array of cephalothoracic swellings. In particular the paired antennary swellings are each composed of about five fused lobes forming an almost triangular protuberance  but neither possesses well developed paired posterior swellings , also lacks posterior swellings although it does possess paired lateral swellings ; both species have a similar array of antennary, maxillary and posterior swellings, both have the neck longer than the trunk , and the posterior processes are shorter than the trunk in both. There are, however, significant differences in the relative development of the cephalothoracic swellings. In T. elongatus the paired maxillary swellings are attached to a “larger central swelling consisting of 2 lateral knobs and 1 median knob”   MAGNT Reg. No. NTM Cr0179497; paratype female intact but lacking egg sacs, from R. acutus , Reg. No. NHMUK 2022.178; headless paratype female, trunk bearing paired egg sacs, from R. acutus , NTM Cr0179498; headless paratype female, trunk bearing paired egg sacs, from R. acutus , NTM Cr0179499.Holotype female, intact with one missing egg sac, from LSID urn:lsid:zoobank.org:act:C53693BF-0A95-4CAB-90EF-9096D0745A32Transformed adult female. Body Fig. A comprisNeck slender, cylindrical with smooth margin; expanded anteriorly at junction with cephalothorax; expanding gradually posteriorly to merge with trunk. Neck longer than trunk, mean 1.75 times, with a range of 1.70 to 1.80 times longer (n = 2). Trunk about 4.4 times longer than wide, slightly compressed dorsoventrally and increasing in width towards maximum at posterior end; bearing paired lateral swellings near posterior end. Abdomen incorporated into trunk; bilobed posteriorly Fig. C, 4C. TrCephalothorax bulbous bearing pair of antennary swellings anteriorly on ventral surface Fig. B. PairedAntennule not observed. Antenna Fig. D biramouMale unknown.Tripaphylus.Etymology. The species name, dippenaarae, honours Susan Dippenaar for her major contributions to our knowledge of the diversity of T. elongatus and T. squidwardin. sp. All three species have a similar array of antennary, maxillary and posterior swellings on the cephalothorax, the neck is longer than the trunk , and the posterior processes are shorter than the trunk. The new species differs from T. elongatus in the relative development of the cephalothoracic swellings. In T. elongatus the paired maxillary swellings are attached to a “larger central swelling consisting of 2 lateral knobs and 1 median knob” (Dippenaar, T. elongatus (see Dippenaar, This new species is similar to T. squidwardin. sp. but these two species differ slightly in body proportions: in T. squidwardin. sp. the neck is a mean of 1.48 times longer than the trunk whereas in T. dippenaaraen. sp. the neck is a mean of 1.75 times longer than the trunk. However, the range of values for T. dippenaaraen. sp. falls within the range found in T. squidwardin. sp. This plus the difficulty in determining the precise boundary between neck and trunk makes this difference unreliable for species discrimination.The new species is most similar to T. squidwardin. sp. the maxillary swellings are about one third of the size of the ventrolateral swellings whereas in T. dippenaaraen. sp. the maxillary swellings are only slightly smaller than the ventrolateral swellings. In addition, the paired ventrolateral swellings are separated along ventral midline by a central ridge in T. squidwardin. sp. but by a well-defined median groove in T. dippenaaraen. sp.There are additional differences between these two new species in the precise arrangement of the lobes on the ventral surface of the cephalothorax. The most striking difference is the relative size of the maxillary swellings and the ventrolateral swellings: in


Escherichia coli and yeast Saccharomyces cerevisiae strains are also being engineered to have cell surfaces displaying hydrolytic enzymes, which holds much promise for near-term scale-up and biorefinery use. Looking forward, future advances to achieve economically feasible production of lignocellulosic-based biofuels with special focus on designing more efficient metabolic pathways coupled with screening, and engineering of novel enzymes.Combating climate change and ensuring energy supply to a rapidly growing global population has highlighted the need to replace petroleum fuels with clean, and sustainable renewable fuels. Biofuels offer a solution to safeguard energy security with reduced ecological footprint and process economics. Over the past years, lignocellulosic biomass has become the most preferred raw material for the production of biofuels, such as fuel, alcohol, biodiesel, and biohydrogen. However, the cost-effective conversion of lignocellulose into biofuels remains an unsolved challenge at the industrial scale. Recently, intensive efforts have been made in lignocellulose feedstock and microbial engineering to address this problem. By improving the biological pathways leading to the polysaccharide, lignin, and lipid biosynthesis, limited success has been achieved, and still needs to improve sustainable biofuel production. Impressive success is being achieved by the retouring metabolic pathways of different microbial hosts. Several robust phenotypes, mostly from bacteria and yeast domains, have been successfully constructed with improved substrate spectrum, product yield and sturdiness against hydrolysate toxins. Cyanobacteria is also being explored for metabolic advancement in recent years, however, it also remained underdeveloped to generate commercialized biofuels. The bacterium   The Cupproducts . Similar ethanol .Methanotrophic bacteria are also an important source of bioproducts for biofuel production. Systems metabolic engineering of such bacterium has led to the advancement in methane-based bio-manufacturing of biofuels ,125. Som4.2.Cyanobacteria are the photosynthetic prokaryotes with more potent photosynthetic metabolomics than land plants. This metabolomics is advantageous to make them potential cell factories, which can efficiently produce biofuels and bio-based chemicals through sequestering of carbon. Also, its well-tractable physiology and more specific growth requirements attract attention to be engineered for industrial purposes .Synechocystis and Synechococcus for improved ethanol production [Cyanobacteria are a good choice for solar-powered bioethanol production due to the presence of ethanologenic pathway . Consequoduction ,129. Sevoduction .Synechocystis PCC 6803 strains were constructed through induced expression of four Calvin-Benson-Bassham (CBB) cycle enzymes, i.e., transketolase (TK), ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO), aldolase (FBA), and fructose-1, 6/sedoheptulose-1, 7-bisphosphatase (FBP/SBPase) coupled with ethanol synthesis enzymes PDC and ADH [Synechocystis PCC 6803 was increased by 2-9-folds by combined expression of multiple native CBB enzymes [S. elongatus PCC7942 strain was developed through induced expression the ictB, ecaA, and groESL, and pdc-adhII genes [2 (25%), SO2 (80–90 ppm) and NO (90–100 ppm).In recent research, recombinant  and ADH . The eng enzymes . More reII genes . The engSynechocystis and Synechococcus were recently constructed through the introduction, overexpression, and evaluation of different genes related to isobutanol and 1-butanol biosynthesis [Synechocystis PCC 6803 strain was developed through the introduction of the genes kdc (keto-acid decarboxylase) and adh under the control of the CcaS/CcaR system. The enhanced strain was able to produce 238 mg/l of isobutanol and 75 mg/l of 3-methyl-1-butanol under red and green light illumination in 5 days [Synechococcus PCC 7002 significantly enhanced the photosynthetic production of 2-methyl-1-butanol and isobutanol [S. elongatus revealed that enhanced isobutanol production was caused by lipid degradation with the increase in NADH. The engineered strain proved to be a practical and feasible system for cost-effective isobutanol production [S. elongatus PCC 11801 has recently evolved in laboratory conditions through adaptive tolerance to higher concentrations of butanol [Recent efforts have also been directed towards engineering cyanobacteria for the production of isobutanol and 1-butanol. Numerous recombinant strains of ynthesis ,136. Recn 5 days . In a diobutanol . Also, moduction . Another butanol . NotwithSynechocystis has recently been metabolically engineered to produce 1-Octanol, one of the emerging precursor molecules for high-value product formation, including fuel. The most efficient computational tools have been proved to be useful in determining appropriate cleavage sites of thioesters in the above cyanobacteria, which further play an important role in selecting 1-octanol [Synechocystis sp. PCC 6803 has been enhanced by the introduction of the α-ketoisocaproate dioxygenase gene from Rattus norvegicus (RnKICD), which resulted in optimization of the isobutene pathway [Synechocystis sp. PCC 6803 has been achieved by Rodrigues and Lindberg [The -octanol . Similar pathway . Also, tLindberg .2 yield by modulating both nitrogenase and hydrogenase-based H2 production systems [2 production at the industrial scale. In addition, large number of cyanobacteria are naturally capable of producing biohydrogen, which can be mainly synthesized by: (a) the action of bidirectional or reversible hydrogenase and (b) as a by-product of nitrogen fixation via nitrogenases . Many no systems ,148, but4.3.Saccharomyces cerevisiae has been the promising host for ethanol production from the earliest time, it cannot efficiently utilize the sugar polymers, such as xylose, and cellobiose available in LCB [Yeasts, including native, thermotolerant, halophilic, etc., have attracted much attention as cell factories convert lignocellulose to biofuels and bio-products. The large spheroidal cells, simple bioprocessing, minimal nutritional requirements, and better inhibitor tolerance relative to many bacteria and cyanobacteria can expand this scenario . Althouge in LCB . Also, ge in LCB .S. cerevisiae strain through combinatorial CRISPR–Cas9-mediated rational and evolutionary engineering. This isomerase-based xylose-fermenting strain, named XUSE, demonstrates the efficient conversion of xylose into ethanol with a high yield of 0.43 g/g and exhibited simultaneous fermentation of glucose and xylose with negligible glucose inhibition. Significantly, higher ethanol yields were also achieved by engineering the high osmolarity glycerol pathway [S. Cerevisiae [Several metabolic engineering strategies to address the issues mentioned above have been undertaken and implemented to yeast in the last couple of years . For ins pathway . Recentlrevisiae .Figure Aspergillus niger) and Xut3 (from Scheffersomyces stipitis), were successfully introduced into yeast to maximize xylose uptake [Neurospora crassa was found suitable for improved xylose transportation and glucose-xylose co-utilization [S. cerevisiae. The increase of ethanol production from xylose compared to that of parental strain confirms their involvement in regulating xylose growth and fermentation.Studies also focused on transporter and transcription factor engineering to develop more powerful ethanol-producing strains as yeast lacks xylose-specific transporters. Some novel xylose transporter, such as XltA , is also being exploited to engineer xylose metabolism. Overexpression of these native transporters with xylitol dehydrogenase improves dxylose utilization and metabolism [Y. lipolytica resulted from 14-folds increased lipid titer [Y. lipolytica strain through CRISPR–Cas9-mediated rational and evolutionary engineering. This isomerase-based xylose xylose-utilizing strain, YSXID, produced 12.01 g/L lipids with a maximum yield of 0.16 g/g, the highest ever reported, from lignocellulosic hydrolysates. In the oleaginous yeast domain, Rhodosporidium toruloides can also utilize carbon sources (apart from sugars) available in lignocellulosic hydrolysates, such as organic acids lignin-derived phenolic compounds [R. toruloides and reported high lipid yield (0.179 g/g) with non-detoxified lignocellulosic hydrolysates. Increased lipid production was also observed in R. Toruloides strains by overexpressing gene encodes Δ9 fatty acid desaturase (Δ9FAD), which synthesize palmitoleic and oleic acids [R. toruloides strains.Prompted by the accomplishments with ethanol producer oduction . These yoduction . The oletabolism . This sptabolism ,164. Theid titer . Yook etid titer  developeompounds ,168. Somompounds  implemenic acids . Recentlic acids  reportedS. cerevisiae, has been extensively improved through several metabolic and evolutionary engineering approaches [S. passalidarum and S. stipites to fermentation inhibitors has not been as extensively studied as that of S. cerevisiae. Although their robustness relies heavily on the type and level of inhibitors present in the hydrolysates [Y. lipolytica to lignocellulosic fermentation inhibitors has also been improved through metabolic engineering approaches [In addition, the robustness of yeast strains against toxic fermentation inhibitors  present in lignocellulosic hydrolysates is crucial for the sustainability of biofuels. The robustness of model yeast, proaches . The tololysates ,174, theproaches ,176. Theproaches . The eng4.4.Mortierella, Mucor, and Aspergillus Cunninghamella [Mucor circinelloides [Ashbya gossypii mainly used for industrial production of riboflavin, is being engineered for increased production of lipid droplets also. Lipid production can be increased by providing lipid precursor and blocking the other competing pathways of lipid accumulation. A knock out in AgPOX1 gene resulted in blocking of β-oxidation pathway and increase the yield  of total lipids, additionally the culture medium was supplied with 1% glucose and 2% oleic acid to prevent the above pathway completely [Mortierella, Aspergillus, and Cunninghamella. In the form of lignocellulosic hydrolysate substrate, mild sulphuric acid treated wheat straw provided sufficient xylose, on which Mortierella isabelline can grow and accumulate high amount of lipid (39.4%) [Mucor circinelloides has been genetically modified. The β- oxidation pathway was modified by integrating heterologous acyl-ACP thioesterase into Fatty acid synthetase complex with a sequential knockout of acyl-CoA thioesterase and/or acyl-Co-A oxidase genes. A total increase in accumulation of medium chain fatty acids was reported around 47.45% in compare to wild strain, which can accumulate only 2.25% [Unsaturated fatty acids are desirable starting material for production of biodiesel. Ascomycota and Mucoromycota are major phyla comprising of oleaginous filamentous fungi capable of production of single-cell oils approximately 15–36% cell dry weight. Single-cell oils rich in monounsaturated fatty acids and saturated fatty acids can be utilized for production of third generation biodiesel. So, lots of work has been done to optimize the culture conditions for maximum production of single-cell oils from ghamella . Severalelloides  Ashbya gmpletely . In the  (39.4%) . The funly 2.25% ,183.Candida rugosa, Rhizomucor miehei, etc., through the immobilization of these strains in nanostructures [Trichoderma spp. and Aspergillus spp. are main source of cellulase enzyme production at industrial scale. T. reesei has higher cellulase content but due to lack of β-glucosidase production co-culture with A. phoenicis gave a 2.5-fold increase β-glucosidase production [Penicillium funiculosum, PfMig1 88, was successfully evaluated to enhance the saccharification of sugarcane bagasse [Nanostructures are very much important for immobilization of various fungal strain in packed bed reactors, to improve the yield of hydrolyzing enzymes. More than 90% yield of biodiesel was reported in ructures . Besidesoduction . In a di bagasse .5.S. cerevisiae, is the major mainstay of this approach. Its surface display was first explored to develop ‘arming yeast’ that can be a good platform for self-immobilized biocatalysts [P. pastoris and Y. Lipolytica [Microbial cell surface engineering is an innovative technique to endow novel functions on host cells through displaying functional proteins or enzymes on the cell surface . This apatalysts . Soon afpolytica ,190. In polytica . It has polytica ,195. Subpolytica .Figure S. cerevisiae. They obtained 3-fold higher ethanol production than a similar yeast consortium secreting only the three cellulases on phosphoric acid swollen cellulose. Similarly, enhancement of hydrolytic activity with improved ethanol titer has been reported while a bi-functional minicellulosome was accomplished on S. Cerevisiae [P. pastoris to display minicellulosomes. The incubation of the protein-displaying yeast with three recombinant cellulases, including endoglucanase, an exoglucanase, and a β-glucosidase fused with a carbohydrate-binding module led to a cellulosome displaying P. pastoris able to direct conversion of carboxymethyl cellulose to bioethanol, observing an impressive ethanol titer of 5.1 g/L. The surface display engineering approaches employed on various microbial consortiums have been attempted to hydrolyze other lignocellulosic components, including xylan and lignin [Interestingly, certain microbial clostridia can produce cellulosome, a complex of scaffolding proteins that requires multiple enzymes for efficient hydrolysis. Goyal et al.  developerevisiae . A cellurevisiae  directlyrevisiae  engineerd lignin . Table 56.in vivo monitoring of the target compounds [Quantification and regulation of the metabolic flux and metabolic pathway are crucial for optimizing the microbial biofuel production processes . Biosensompounds . Severalompounds , isoprenompounds , fatty aompounds , butanolompounds , and fatompounds . More adompounds . There aompounds ,213,214.ompounds ,216.Fig7.Z. mobilis or their allied species express xylanases that enable pentose sugars’ catabolism with improved ethanol production [Synechococcus 7942 and Synechocystis 6803 systems. Algenol has filled various patent starting from 1998, publication number US6306639B1, followed by US8163516B2 (filed 9 February 2009), which was in itself first patent showing increased ethanol yields via overexpression of an alcohol dehydrogenase [Synechococcus 7002 framework [Protection of intellectual property (IP) related to the strategies and methods to improve biofuel production by modifying microorganisms is under intense development. Several recombinant oduction ,218 and oduction  have beeoduction  restrainoduction  and thatoduction ,222 haveoduction . This paoduction  that desrogenase  Further,rogenase . The Jouramework .Table 6.8.in vitro [Recombinant microorganisms have opened new alternatives for cleaner and more efficient production of various metabolites including biofuels. Still, high cost, low efficiency of raw material conversion and unintentionally release of genetically modified microorganisms in the environment have limited the use of bioprocess using living cells and cell-free metabolic engineering becoming popular to harnesses the metabolic activities of cell lysates in vitro . Similarin vitro . The genin vitro . Though in vitro ,232.Environmental risk assessment of various genetically modified organisms (GMO) has been done time to time  but stil9.S. cerevisiae, are already being engineered and commercialized to ferment simple sugars to isobutanol and other alcohols. Some non-conventiona,l such as S. passalidarum, P. stipites, etc., and a few oleaginous yeasts, i.e., Y. lipolytica, R. toruloides are being engineered to produce both bioethanol and biodiesel. Yeast strains engineered to surface display hydrolytic enzymes appear to be much more promising and have a broader prospect for commercial-scale bioethanol production directly from LCB in upcoming years. The bacterium Z. mobilis, E. coli, and other thermophiles are being engineered to ferment the xylose and could be used in commercial processes for biofuels production from lignocellulosic hydrolysates. Cyanobacteria and other photosynthetic bacteria also seem to generate commercializable biofuels in the future potentially. Although metabolic engineering is a well-established strategy for transforming microorganisms into efficient cell factories, there is a high possibility that novel robust industrial phenotypes could emerge from the coupling of more database mining, omics technologies, and advanced genome engineering strategies. Futuristic, metabolic engineering of microorganisms to discover new enzymes catalyzing unknown reactions, engineered enzymes with enhanced performances, and new synthetic pathways designed for higher metabolic flux towards target biofuels and other value-added products may enable the production of more sustainable biofuels.Through the application of metabolic and bioprocess engineering approaches, numerous challenges associated with the production of lignocellulosic biofuels have been successfully revolutionized during the past few years. In brief, improvements in the biosynthesis of polysaccharides, lignin, and lipids in the lignocellulosic biomass have led to limited success, and further progress is required for sustainable biofuels. Metabolic pathways of microbial strain from diverse domains, such as yeasts, bacteria, and cyanobacteria have been successfully engineered to produce bioethanol from lignocellulose with a high yield near its maximum theoretical yield (0.51 g/g glucose). Yeasts, mainly


Aspergillus fumigatus is an opportunistic airborne fungus that causes severe invasive aspergillosis in immunocompromised patients. Zinc is an essential micronutrient for the growth of A. fumigatus and even for all microorganisms. An increasing number of studies have reported that fungal zinc acquisition ability plays a key role in fungal survival in hosts with an extremely zinc-limited microenvironment. The ability to fight scarcity and excess of zinc are tightly related to fungal virulence and may be used as new potential targets. Because the regulation of zinc homeostasis is important, a thorough understanding of the functional genes involved in the regulatory network for zinc homeostasis is required for fungal pathogens. The current mini-review summarized potential zinc homeostasis regulators in A. fumigatus and classified these regulators according to localization and function, which were identified or predicted based on A. fumigatus or deduced from homologs in model yeasts. Future perspectives for zinc homeostasis regulators as potential antifungal targets to treat invasive aspergillosis are also discussed. Aspergillus fumigatus is an opportunistic and saprophytic filamentous fungal pathogen that proliferates in the lungs of immunocompromised patients and causes invasive pulmonary aspergillosis, which is an infectious disease with a fatality rate of up to 95% [A. fumigatus isolates have been found in many countries in recent years [p to 95% ,2,3,4,5.p to 95% . Howevernt years ,8,9. TheA. fumigatus relies on its pathogenicity and host immune capacity, but its pathogenicity is multifactorial and sophisticated [2+ is closely related to the pH of the environment, and it readily forms complexes with other compounds as the pH increases [2+ in the host body at physiological pH (7.3–7.4) is very low because most of the zinc in intracellular and extracellular fluids is firmly bound to zinc-binding proteins [2+ and Mn2+ utilization and inhibit microbial growth [A. fumigatus to promote the search for new therapeutic targets and the development of antifungal drugs to fight fungal pathogens.The virulence of sticated . The hossticated ,12,13. Zsticated ,15,16. Tsticated ,21,22,23sticated ,25,26. Tncreases . Therefoproteins ,29. The proteins . When thl growth ,32,33. Zl growth . TherefoA. fumigatus primarily comes from results in the model yeast Saccharomyces cerevisiae [S. cerevisiae [S. cerevisiae and the Irt1 protein of Arabidopsis thaliana, and it belongs to the solute carrier protein SLC39A family [S. cerevisiae [S. cerevisiae ZIP transporters, Zrt1 (zinc-regulated transporter) was first identified as a high-affinity transporter protein for zinc uptake under zinc-deficient conditions [A. fumigatus  has not been demonstrated. Therefore, their roles in zinc transport in A. fumigatus must be further investigated.Zinc transporters on organelles also play important roles in zinc homeostasis. and Atx2 . Zrt3 isand Atx2 . Yke4 isand Atx2 . The ZIPand Atx2 ,57. HoweS. cerevisiae indicate that this process depends on Zrc1 and Cot1 zinc importers on the vacuolar membrane [S. cerevisiae, A. fumigatus has three putative vacuolar CDF transporters, ZrcA (AFUB_092140), ZrcB (AFUB_098870) and ZrcC (AFUB_030200). Of these three CDF transporters, ZrcC has the highest homology to the yeast Zrc1/Cot1 protein, while ZrcA responded to high-Zn stimulation [A. fumigatus [Another transporter family that plays an important role in zinc homeostasis is the cation diffusion facilitator (CDF) family, which contributes to the transport of excess metals from the cytoplasm to intracellular organelles ,59. The membrane ,61,62,63mulation ,64. A Zrumigatus . HoweverA. fumigatus, ZrgA (AFUB_011560), MscA (AFUB_000580) and MmtA (AFUB_057390), respectively, but their functions as CDF family proteins have not been investigated. In contrast to S. cerevisiae, there are two other unique CDF family proteins, MtpA (AFUB_013980) and MtpB (AFUB_097080), in A. fumigatus, whose functions have also not been investigated. Therefore, further studies on the function of CDF protein family members are required to supplement and perfect the zinc detoxification mechanism of A. fumigatus.Two other CDF family members, Zrg17 and Msc2, localize to the ER membrane and compose a heteromeric complex that imports zinc into the endoplasmic reticulum for proper protein processing ,66. The S. cerevisiae is sensed by the zinc-responsive transcription factor Zap1, which activates Zrt1/2 of the plasma membrane and Zrt3 of the vacuolar membrane in response to zinc starvation under zinc-limited conditions, regardless of the ambient pH [S. cerevisiae, A. fumigatus zinc-responsive transcription factor ZafA is a potential homolog of S. cerevisiae Zap1 that governs the expression of zinc uptake genes [S. cerevisiae, the expression of the zinc transporters in A. fumigatus, ZrfA, ZrfB and ZrfC, is influenced by the environmental pH and the zinc concentration. Therefore, A. fumigatus has evolved delicate mechanisms to effectively obtain the necessary zinc from acidic or alkaline environments. Under acidic zinc-limiting conditions, the A. fumigatus transcription activator ZafA combine with zinc response (ZR) motifs in the zrfA and zrfB promoters to induce their expressions [zrfC promoter, but ZrfC expression is inhibited by another transcription factor, PacC [zrfA was partially inhibited under alkaline zinc-limiting conditions, and the expression of zrfB was strongly inhibited by the transcriptional regulator PacC [zrfC [A. fumigatus has evolved a more refined regulatory mechanism to regulate zinc homeostasis compared to yeast.Zinc homeostasis in fungi is primarily regulated at the transcriptional level via zinc-responsive transcription factors. Zinc starvation in bient pH ,70. Simike genes ,72. Unlior, PacC . PacC isor, PacC ,74. The cC [zrfC . In summA. fumigatus is involved in copper detoxification and plays an important role in zinc detoxification [A. fumigatus is exposed to high Zn conditions, AceA significantly induces the expression of the plasma membrane efflux pump CrpA and the vacuolar transporter ZrcA. Although AceA also induces the expression of the metallothionein CrdA in response to high Zn, A. fumigatus primarily uses CrpA and ZrcA, rather than CrdA, as zinc-resistance mechanisms [A. fumigatus. Therefore, the identification of the binding motifs of AceA will be the priority of future studies in A. fumigatus.Although zinc is required for fungal growth, excess zinc is potentially toxic to fungal cells. Therefore, fungi also have high levels of zinc-responsive transcription factors that respond to excess zinc. The copper-responsive transcription factor AceA in fication . Howeverfication . When A.chanisms . HoweverA. fumigatus in alkaline and extreme zinc-limited environments [A. fumigatus  and zinc transporters (import/export), which are important in the virulence of A. fumigatus [A. fumigatus since the ΔzrfC mutant showed significantly reduced virulence in an immunocompromised murine model of invasive aspergillosis (IA) [A. fumigatus. The role of ZrfC protein in fungal virulence primarily depends on its special long N-terminus on the extracellular side of the membrane to obtain Zn directly from the lungs, which is not present in the acidic transporters ZrfA or ZrfB [zrfA, zrfB and zrfC are highly induced in a ZafA-dependent manner in a zinc-limited environment, ZafA is also considered a virulence factor for A. fumigatus [S. cerevisiae and induced the vacuole transporter Zrt3 to mobilize stored zinc in response to zinc deficiency [Aspergillus species, but its role in virulence as a zinc transporter on vacuoles in A. fumigatus has not been investigated. Similar to ZafA, another transcription factor PacC was necessary for the virulence of A. fumigatus because PacC deletion significantly reduced virulence [crpA and zrcA, and it is required for A. fumigatus virulence [A. fumigatus primarily by regulating the detoxification of copper by crpA, but it is not known whether the involvement of AceA in the detoxification of Zn contributes to virulence. However, it has been shown that host macrophages provide a mechanism of zinc poisoning against Mycobacterium tuberculosis [Since host lung tissue provides a slightly alkaline zinc-limited environment to inhibit microbial growth, fungal pathogens have a highly coordinated mechanism to regulate zinc homeostasis to prevent zinc deficiency and thus promote their survival. As mentioned previously, umigatus ,71,73. Asis (IA) . Triple  or ZrfB . Becauseficiency ,70. ZrtFirulence . AceA isrculosis . This meA. fumigatus as a monotherapy or in combination, and clinical mortality remains high [A. fumigatus-specific metabolic pathways are necessary as an alternative to the proteins targeted by classic antifungals. Since A. fumigatus has evolved an effective zinc-acquisition mechanism to counteract zinc limitation, we propose a therapeutic strategy based on preventing zinc acquisition to inhibit A. fumigatus growth. The zinc uptake by A. fumigatus in the slightly alkaline environment of the host is primarily mediated by the zinc importer ZrfC [A. fumigatus in the host. ZrfC primarily relies on a specific N-terminal uptake of extracellular zinc, and there is no homolog in humans [A. fumigatus is essential for the regulation of zinc homeostasis and virulence, and A. fumigatus uses ZafA to activate zinc importers ZrfA, ZrfB and ZrfC, which prevents zinc deficiency [ΔzafA mutant exhibited lower virulence than the ΔzrfC mutant in an immunocompromised mouse model, which suggests that ZafA plays a major role in the virulence of A. fumigatus [2H2-type zinc fingers of transcription factors in the host, the inhibition of ZafA should be based on a specific blockade of its transcriptional activation domain rather than interference with its C2H2-type zinc finger-dependent DNA binding domain.With the recent increase in drug-resistant strains, antifungal drugs used to treat fungal pathogens are not sufficiently effective for inhibiting the growth of ins high . Therefoter ZrfC . Therefon humans . Thus, aficiency ,72. Notaumigatus . SimilarRecent academic research and biomedical companies have effectively used fragment-based drug discovery to investigate selective inhibitors against highly conserved effective targets . AlthougA. fumigatus infection may provide a promising therapeutic target. In future studies, new compounds specifically blocking the regulation of zinc homeostasis in A. fumigatus could be tested for potential as antifungals. Moreover, the molecular characterization of fungal-specific zinc-responsive transcription factor ZafA and zinc importer ZrfC must be further explored and used as potential drug targets for screening novel antifungal drugs through fragment-based drug discovery or other drug-screening strategies.Taken together, the regulation of zinc homeostasis required for


Culex quinquefasciatus, ranging from 120 to 360 min. Petitgrain oil showed the highest complete-protection time from bites of Aedes aegypti for 270 min. Peppermint oil also exhibited complete-protection time from bites of Anopheles dirus for 180 min. When two highly effective essential oils were combined to potentially prolong mosquito bite protection, the binary combinations of petitgrain with other essential oils (basil and coriander) exhibited antagonistic effects and provided complete protection against A. aegypti bite for 150 min. On the other hand, the sage and patchouli oil combination showed more repellent activity from A. dirus bite for 270 min than an individual oil. The binary combination efficiently increased or decreased protection time.The repellent efficacy of ten essential oils was measured against all three-mosquito species using the arm-in-cage approach to determine the duration of protection. All essential oils showed high complete-protection time against A. aegypti, A. dirus and C. quinquefasciatus using the arm-in-cage method. Petitgrain oil was the most effective against A. aegypti (270 min). Peppermint oil was the most effective against A. dirus (180 min). Interestingly, all single oils had attributes of repellency against C. quinquefasciatus . Moreover, we integrated their binary combinations of highly effective essential oils against A. aegypti and A. dirus to potentially increase the protection time. A 1:1 combination of petitgrain/basil, petitgrain/coriander, basil/coriander and basil/sage reduced the median complete-protection time of 150 min for A. aegypti; a combination of sage and patchouli oils prolonged the median complete-protection time of 270 min for A. dirus. Combining essential oils effect protection time from these two mosquito species.Mosquito repellents reduce human-vector contact of vector-borne diseases. We compared the repellent activity of 10 undiluted essential oils  against  Aedes, Culex [Anopheles mosquitoes, respectively. In Thailand, lymphatic filariasis was studied in 23,477 targeted immigrants from 2002 to 2017; the findings revealed that 0.7% of them became seropositive on average [Dengue and malaria are mosquito-borne diseases that are widespread in tropical and sub-tropical regions. The primary vectors of dengue, filariasis and malaria are s, Culex ,2 and An average .N,N-diethyl-m-toluamide (DEET) is a synthetic chemical insect repellent that is effective against mosquitoes but can penetrate the skin and cause adverse health effects in humans [Aedes, Anopheles, or Culex genera. These include Ocimum basilicum (basil), Citrus bergamia (bergamot), and Coriandrum sativum (coriander) [Personal protection is the simplest method of preventing mosquito bites and reducing mosquito-borne diseases and includes window and door screens, long-sleeved shirts, long trousers, mosquito bed nets and insect repellents . N,N-dien humans ,8,9. Then humans . Multipln humans . Essentiriander) ,13,14.A. aegypti, A. dirus and C. quinquefasciatus, as well as their binary combinations against A. aegypti and A. dirus using the arm-in-cage method under laboratory conditions to identify effective candidates for plant-based mosquito repellents.Efforts to improve the efficacy of repellents have involved the addition of fixatives such as paraffin, vanillin and coconut oil. Another option would be to combine essential oils for an additive or synergistic effect . In ThaiThe 10 essential oils were selected from the literature on their mosquito-repellent activity, edibility and non-toxicity to humans . The 100v/v). Each combination was specific for each mosquito species listed in The essential oils that had  and C. quinquefasciatus (Nonthaburi strain), and the forced-mating A. dirus (Kao Mai Kaew strain) laboratory-reared females were maintained at the Insecticide Research Unit at the Department of Medical Entomology, Faculty of Tropical Medicine, Mahidol University. Eggs were hatched in plastic trays. After hatching, 200 larvae were reared in plastic trays in 1500 mL of dechlorinated water and fed daily on fish food powder . Pupae were transferred to plastic cups and placed inside a cage (20 × 20 × 30 cm3) for emergence. Adult mosquitoes were fed 5% sugar solution on cotton wool, which was changed once a week. All larvae and adult mosquitoes were colonized and maintained under controlled insectary conditions . Both sexes of mosquitos were maintained in the cage to allow for mating [A. aegypti and C. quinquefasciatus are free-mating in the cage, while A. dirus requires forced mating. The non-blood-fed female mosquitoes, 5–7 days old A. aegypti and A. dirus, and 10–12 days old C. quinquefasciatus were isolated and sugar fasted for 12 h before testing but had access to limited water. Female mating or unmating had no effect on blood feeding [The free-mating r mating . Only A. feeding .C. quinquefasciatus were more active in seeking hosts than 5–7-day-old, resulting in more than 10 bites on the control arm within 3 min.The age of female mosquitoes was considered in this study in accordance with WHO recommendations  that theFive male and female volunteers were 20–65 years old, healthy, with no history of an allergic reaction to mosquito bites. We excluded women who were pregnant and individuals who were sensitive to essential oils or taking daily medication. Volunteers did not use fragrance and repellent products for 12 h before the experiment and during testing. Volunteers were not tobacco users or at least had refrained from tobacco use for 12 h before and during testing. Volunteers were interviewed and instructed in the methodology. Each volunteer signed an informed consent form.A. aegypti, A. dirus and C. quinquefasciatus using the arm-in-cage technique modified from the standard World Health Organization method [A. aegypti and A. dirus females, and 250 fasted aged 10–12 days C. quinquefasciatus were selected at random and placed in a standard cage (30 × 30 × 30 cm3) and rested for 1–2 h before the experiment. Tests were conducted by exposing the control and tested forearms in the test cage containing mosquitoes. The control arm was exposed before the treatment arm. If at least 10 mosquitoes landed on the control arm within 3 min, the repellency test was continued. After 30 min post application, the tested arm was exposed for 3 min, then withdrawn for 30 min, and then exposed again. The experiment was stopped when the second mosquitoes landed on the test area and recorded as the complete-protection time for that subject [A. aegypti should be at least 120 min [A. aegypti and A. dirus were exposed from 8:00 to 15:00 h, and C. quinquefasciatus from 15:00 to 22:00 h.Undiluted essential oils were evaluated for repellency against female n method  and Junkn method . Each es subject . The pro 120 min , which iC. quinquefasciatus prefers to bite at night and is less anthropophilic [pophilic . Then, 2The complete protection time of each volunteer was recorded. These data were non-normally distributed and were small samples; therefore, the differences in the protection time and the type of essential oil were analyzed by using the non-parametric Kruskal–Wallis test. If the ANOVA was significant, Dunn’s multiple comparisons were performed with the Benjamini–Hochberg correction at a critical level of 0.05 .A. aegypti by the arm-in-cage method  among the individual essential oils. The results of pairwise comparisons of individual essential oils  among the individual essentials. The complete results of pairwise comparisons of individual essential oils against A. dirus were provided in The median protection time of 10 undiluted tested essential oils against nditions B. When cp = 0.001, df = 10) among the individual essential oils. The results of pairwise comparisons of individual essential oils against C. quinquefasciatus were provided in Followed by FDA Thailand requirements, the repellent time against mosquitoes should be more than 120 min; all 10 tested essential oils showed high levels of protection C, with mA. aegypti at a 1:1 (v/v) ratio to evaluate whether they exerted additive or synergistic effects . The combinations were not significantly different in their efficacy against A. aegypti . Since the analysis of variance was not significant, the post-hoc comparisons were not performed.We mixed the four essential oils  in two-oil combinations against  effects . All com effects . A mediaA. dirus at a 1:1 (v/v) ratio to evaluate whether they exerted additive or synergistic effects . However, the combined essential oil prolonged repellency against A. dirus over that of individual oils. Because the non-parametric Kruskal–Wallis test was not significant, the pairwise comparisons were not calculated.The five essential oils  were blended into two-oil against  effects . All com effects . CombiniA. aegypti, A. dirus and C. quinquefasciatus to some degree in the human arm-in-cage assay. Few essential oils examined have higher repellent efficacy than citronella, the well-known natural mosquito repellent in Thailand [A. aegypti. Conversely, the FDA Thailand does not prescribe for other mosquito species, and when considering the repellent effect of citronella oil on A. dirus, the protection time of 90 min was revealed, which was consistent with previous studies [C. quinquefasciatus, all tested essential oils showed repellent potential with protection time higher than 120 min. Petitgrain and basil oil exhibited the highest repellency, with the protection time of 360 min equaled to citronella. This was in concordance with earlier studies [C. quinquefasciatus. Combining them to improve repelling duration is unnecessary in this study. However, this is a significant benefit that we will study further by integrating it with other product and packaging technologies, featuring a prototype product that is reliable and comfortable for users. Above all, we directly targeted further development of plant-based repellents for anti-mosquito vectors in dangerous vector-borne diseases, urban (A. aegypti), forest and jungle areas (A. dirus).Our research clearly revealed that some essential oils studied were effective against Thailand . The FDA studies ,22,23. A studies ,25. We cA. aegypti, in line with previous reports [A. aegypti and are known to contain insect-repelling compounds such as α-terpineol, citral, eugenol, linalool, menthol and estragole [A. aegypti, A. dirus, and C. quinquefasciatus [A. albopictus [A. aegypti and A. quadrimaculatus [Petitgrain oil was the most effective against  reports ,27,28. Estragole ,30,31. Dasciatus ,32. The bopictus . Sage oiaculatus ; its maiaculatus ,34,35.A. dirus (180 min), longer than the 64 min recorded for 10% peppermint oil in soybean oil [A. aegypti for 150 min [A. dirus by essential oils of sage, anise, patchouli and petitgrain. This finding is consistent with previous reports for patchouli against A. dirus [A. stephensi [A. dirus in combination with vanillin [A. stephensi, A. aegypti, and C. quinquefasciatus [The 100% peppermint oil exhibited the highest efficacy against bean oil . A previ 150 min . We alsoA. dirus  and sagetephensi . Patchoutephensi ,38, as wtephensi . Petitgrvanillin . The majvanillin , and it asciatus .Lantana camara flower extract in coconut oil was shown to protect against A. albopictus and A. aegypti without side effects in human volunteers [A. aegypti and A. dirus [A. aegypti was shown to be lower in all six binary combinations than in single oils, suggesting that some components present in each essential oil have antagonistic effects or interact antagonistically with each other [The repellent activity of essential oils depends on their chemical constituents . Camphorlunteers , while aA. dirus . Accordich other .A. aegypti for 150 min, and the combination of sage and patchouli was most effective against A. dirus (270 min). Although no synergistic interaction was found when considering the repellency of oil combinations in repelling A. aegypti, antagonism in the repellent activity of plant essential oils or their main components is not unusual. In particular, as compared to those single oils, the repellent efficacy of the binary blends of sandalwood oil with marjoram, cypress, lemongrass and clary sage oil revealed an antagonistic effect to repel spider mite, Tetranychus urticae [Spodoptera littoralis larvae in 150 of the 435 combinations [C. quinquefasciatus larvae in 74 of the 435 combinations [n. dirus were combined, they improved the median complete-protection time and had a synergistic activity. A similar trend in synergistic effect was found in an increase in the mean protection time against female Stomoxys calcitrans generated by binary combinations of patchouli and tamanu oil (138 min) compared to patchouli oil (41.4 min) or tamanu oil (33.3 min) alone [Anopheles mosquito [We tested binary mixtures of the most effective essential oils for additive or synergistic effects. Petitgrain/basil, petitgrain/coriander, basil/coriander and basil/sage combinations were shown to be efficient against  urticae . Monoterinations  and C. qinations . Interesn) alone . Additiomosquito . TherefoAedes and Culex mosquitoes, for instance, appear to express odorant receptors that respond to DEET but are not expressed in Anopheles [Finally, the different species of mosquitoes have different repellent responses to essential oils . Essentinopheles .C. quinquefasciatus showed an easier repel response with essential oil than A. aegypti and A. dirus, which is similar to other reports [C. quinquefasciatus was sensitive to all 41 essential oils tested, but A. aegypti was tolerable to several oils [C. quinquefasciatus feed on animals and can seek blood meal elsewhere while being rejected by human hosts [A. aegypti and A. dirus are extremely anthropophilic, meaning they more frequently attack humans [In this study,  reports ,60. C. qral oils  due to iral oils . This fian hosts , but A. k humans ,62.C. quinquefasciatus, petitgrain and basil oils being the most potential. The combinations of petitgrain/basil, petitgrain/coriander, basil/coriander and basil/sage were responsible for repelling A. aegypti bite, although the repellent efficiency was lower than that of the individual oils, and sage/patchouli combinations yielded effective and long-lasting protection against A. dirus. These combinations may be candidates for the formulation of safe and effective mosquito repellents. The synergistic repellent effect of the essential oils used in this study may contribute to the development of more effective alternatives to synthetic repellents for human mosquito protection.Essential oil-based repellents are considered safer and more accessible than synthetic chemical repellents and may have a lower probability of resistance development in mosquitoes. Plant-based mosquito repellents can be applied to skin or clothing without affecting non-target organisms or the environment . In the A. aegypti and C. quinquefasciatus. Combining petitgrain/basil, petitgrain/coriander, basil/coriander and basil/sage showed efficacy against A. aegypti, as did a combination of sage and patchouli oils against A. dirus. Field trials should include a study of operational feasibility and dermal toxicity before these essential oil combinations can be developed as alternative and safe insect repellents for individual use to reduce and control vector-borne diseases.Petitgrain, peppermint and basil oil were highly effective against female


Brachybacterium species are ubiquitous Gram-positive bacteria. Here, we report the complete genome sequence of Brachybacterium sp. strain NBEC-018. The strain was isolated from rotten potatoes that were infected with potato rot nematodes (Ditylenchus destructor). The genome sequence will be beneficial to clarify the ecological role of Brachybacterium species. Brachybacterium, which belongs to the family Dermabacteraceae, was first described by Collins et al. in 1988  collected in a farm field in Wuhan, China . The collected potatoes were infected with potato rot nematodes (Ditylenchus destructor), an important plant-parasitic nematode  of 96.22% with Brachybacterium sp. strain SGAir0954 (GenBank accession number CP027295) , 53 tRNA genes, and 9 rRNA genes, as annotated by NCBI PGAP v.6.1 (By using Unicycler v.0.4.8  with defP027295) ; therefoBrachybacterium sp. strain NBEC-018 has been deposited in the GenBank database under accession number CP100650. Sequencing reads have been submitted to the SRA database under accession numbers SRR19995284 and SRR19995286.The genome sequence of