Pomphorhynchus laevis, Neoechinorhynchus agilis, Neoechinorhynchus buttnerae) from four fish species .With the expansion of animal production, parasitic helminths are gaining increasing economic importance. However, application of several established deworming agents can harm treated hosts and environment due to their low specificity. Furthermore, the number of parasite strains showing resistance is growing, while hardly any new anthelminthics are being developed. Here, we present a bioinformatics workflow designed to reduce the time and cost in the development of new strategies against parasites. The workflow includes quantitative transcriptomics and proteomics, 3D structure modeling, binding site prediction, and virtual ligand screening. Its use is demonstrated for Acanthocephala (thorny-headed worms) which are an emerging pest in fish aquaculture. We included three acanthocephalans (The workflow led to eleven highly specific candidate targets in acanthocephalans. The candidate targets showed constant and elevated transcript abundances across definitive and accidental hosts, suggestive of constitutive expression and functional importance. Hence, the impairment of the corresponding proteins should enable specific and effective killing of acanthocephalans. Candidate targets were also highly abundant in the acanthocephalan body wall, through which these gutless parasites take up nutrients. Thus, the candidate targets are likely to be accessible to compounds that are orally administered to fish. Virtual ligand screening led to ten compounds, of which five appeared to be especially promising according to ADMET, GHS, and RO5 criteria: tadalafil, pranazepide, piketoprofen, heliomycin, and the nematicide derquantel.tinyurl.com/yx72rda7.The combination of genomics, transcriptomics, and proteomics led to a broadly applicable procedure for the cost- and time-saving identification of candidate target proteins in parasites. The ligands predicted to bind can now be further evaluated for their suitability in the control of acanthocephalans. The workflow has been deposited at the Galaxy workflow server under the URL The online version contains supplementary material available at 10.1186/s12864-022-08882-1. The global market for antiparasitics, or parasiticides, currently amounts to about seven billion euros per year . Most ofCopaifera [Extracts from garlic , thyme  and diffopaifera  might haopaifera ). Howeveopaifera , 10. Altopaifera ). Well-kopaifera , 12, 13.opaifera , 15. Theopaifera –24. Consopaifera ). Also, opaifera , 27. Addopaifera –30) whilopaifera , 32. ThuPlasmodium falciparum [Ancylostoma duodenale, Ascaris lumbricoides, Brugia malayi, Echinococcus granulosus, Fasciola hepatica, Haemonchus contortus, Necator americanus, Onchocerca volvulus, Ostertagia ostertagi, Schistosoma spp., Strongyloides stercoralis, Taenia solium, Teladorsagia circumcincta, Toxocara canis, and Trichuris trichiura  it is now possible to predict 3D models with much greater precision de novo than before . Neverthlciparum , and addlciparum . In addiiewed in ). These iewed in , 39. ButSus scrofa domestica) and chicken  , pirarucu (Arapaima gigas), Nile tilapia (Oreochromis niloticus), and tambaqui (Colossoma macropum), amongst others [Acanthocephalans are common parasites in the intestinal tract of many mammals, amphibians, birds, turtles, lizards, snakes, and fishes . Depend) e.g., , 51). Th. ThSus s) e.g., , 52, 53), 53Sus st others –57. Heret others , 58. In t others , 59–64.Pomphorhynchus laevis  Monticelli, 1905  [Barbus barbus), a definitive host in which P. laevis matures and reproduces [Anguilla anguilla), in which P. laevis survives [P. laevis specimens from barbel and eel. To enable enhanced specificity of a future control of acanthocephalans, we screened for transcripts which were unique to or at least highly derived in P. laevis compared to diverse non-acanthocephalan species. Moreover, the ideal candidate target had to be readily accessible to drugs. This criterion was approximated by searching proteome data of P. laevis body walls for high-abundance proteins. To increase transferability of the results, protein sequences were checked for matches in new draft genomes of two additional fish-parasitizing species . These were Neoechinorhynchus agilis  Van Cleave, 1916 from Adriatic thinlip mullet (Chelon ramada) and Neoechinorhynchus buttnerae Golvan, 1956 parasitizing above-mentioned tambaqui in South-American limnocultures. Predicted 3D models of the proteins meeting all these requirements were used to screen for drugs that might bind to acanthocephalan target proteins.First genome and transcriptome assemblies for Acanthocephala have lately been published for the Eurasian species cephala) . In the produces , 67, andsurvives  but usuasurvives . For raiP. laevis  from two different host species were used for sequencing, resulting in four pairings of worm sex and host species. Each group included five worm specimens, so possible confounding factors in individual samples should not have affected downstream analysis. The resulting 20 RNA-Seq datasets contained 32.6 million single-end reads (75 bp) on average. Quality processing and mapping to an amended version of the reference transcriptome [Male and female specimens of criptome  was succP. laevis  in a minimum of two out of four pairs of comparisons: (1) female vs. male worms from barbel, (2) female vs. male worms from eel, (3) male worms from barbel vs. eel, and (4) female worms from barbel vs. eel  and in the SwissProt database at an E-value <1e-50 (tblastn/blastp). Correspondingly, agents tailored to these targets should specifically impair acanthocephalans but no other taxa.Mapping with RSEM revealed that the reads from RNA-Seq spread across transcripts of 18,740 genes. For downstream analysis of transcript abundance DESeq2) we only considered genes that had at least 50 mapped transcript reads in each of the 20 samples, thus suggesting ubiquitous expression in  we only P. laevis body-walls, the mass spectra matched 2548 ORFs in the reference transcriptome. Abundances of these proteins  were significantly positively correlated with transcript abundances according to read counts . Thus, higher transcript abundance overall indicated elevated protein abundance. The search for parasitic target molecules was continued with 52 MS-verified proteins .Since transcript abundance does not necessarily correlate with protein abundance , we valins Fig. . As to bp = 3.6 e-07, Student’s t-test; Supplementary Table SThe choice of candidates was supported by the fact that the matching rate between both subsets (52/121 = 0.43) was about three times higher than between the corresponding full lists . In further support of the validity of the approach, the 52 proteins matched several expected properties. Thus, their mean length (485 amino acid residues) was very close to the average in eukaryotes (472 amino acid residues), and clearly exceeded corresponding averages in unicellular species (ca. 300 amino acid residues) . FurtherP. laevis. This prompted us to perform de novo predictions using AlphaFold2 [The above filtering for dissimilar genes ruled out to use structure models of homologous proteins in non-acanthocephalan species as a starting point. In fact, a database search (NCBI) did not reveal a deposited 3D model of a protein structure for any of the 52 candidate target proteins in phaFold2 , which iphaFold2  .Based on the AlphaFold2 models, COACH-D  identifiEight of the eleven putative targets in acanthocephalans were likely to have enzymatic activity according to ECPred, and ten of the target molecules contained PFAM motifs Table , S4. FurPresent results demonstrate the utility of a workflow for identifying putative ligands to target proteins in parasites. The approach is based on genomic, transcriptomic, and proteomic data, followed by 3D structure and binding site prediction, virtual ligand screening, and ADMET property prediction. Applied to fish-parasitizing acanthocephalans, we identified eleven proteins the blocking of which should enable an effective and specific control of these parasites. Five of the ten identified ligands appear to be particularly promising for further testing in vitro and in fish aquacultures.Neoechinorhynchus species as mandatory. This criterion ensured the determination of target proteins that are conserved among fish-parasitizing palaeacanthocephalans and eoacanthocephalans from distant geographic regions (South America and Eurasia). More importantly, one of the species included was N. buttnerae, which is the economically most important acanthocephalan pest species in fish farms. In fact, this species is the major problem in South American limnocultures of fish, decreasing the yield of aquaculture farms by up to 90% [N. buttnerae has direct implications for the practical use of the present results.The filtering workflow was designed to converge on druggable targets in fish-parasitizing acanthocephalans Fig. . One reqp to 90% , 59–64. We consider present protein structure models as reasonably reliable for the prediction of binding sites and subsequent ligand screening. This is because the 3D models were obtained using the two most accurate de novo modeling tools currently available, Alphafold2 and RoseTTAFold , 73, 79.Caenorhabditis elegans life cycle and nematode-specific active binding sites [Another compound for which high affinity was predicted to two of our eleven candidate targets was tadalafil, a phosphodiesterase (PDE) inhibitor Table . Strikinng sites , 86. In ng sites . Such a ng sites . Also, tC. elegans [Anthelmintic potential seems possible for piketoprofen, heliomycin, and pranazepide too, due to their anti-inflammatory, RNA synthesis antagonist, and cholecystokinin receptor type A antagonist activity, respectively –91. For  elegans . Also, w elegans , 13.The development of drugs for parasite control usually takes many years and can easily cause enormous costs for pre-clinical and clinical trials, environmental impact assessment, approval, and the establishment of industrial production. Here, we present a bioinformatics workflow intended to reduce time and cost that is also applicable to non-model parasites for which little functional information is available. The entire workflow includes the identification of candidate targets in parasites and subsequent virtual screening for ligands. Detailed steps are quantitative transcriptome and proteome analyses, prediction of 3D protein structures and binding sites, and virtual database screening for binding compounds. In addition to the novel combination of individual analyses, the approach to the best of our knowledge utilizes for the first time in target identification environmental variation which the parasite is exposed to in definitive and accidental hosts.Application of the workflow to fish-parasitizing acanthocephalans led to the identification of eleven top-ranked candidate target proteins Table . CompounFuture simulations might shed light on the detailed nature of interaction between the candidate targets and ligands mentioned above. Probably, it will be revealing to examine the extent to which the 3D models determined here represent the active protein structure. Moreover, enabling fit induction by the ligand might uncover hitherto hidden binding sites in acanthocephalan proteins , 95. SucN. agilis and N. buttnerae we refer to Supplementary Notes SP. laevis s.l. [N = 20 worms, with 10 specimens  from common barbel and 10 specimens  from European eel. All samples were sequenced as 75 bp single-end reads on an Illumina HiSeq 2500. Raw sequences are available at the EMBL Nucleotide Sequence Database (ENA) repository under accession numbers ERS7302868–87 in project PRJEB47442. Adapter sequences and low-quality parts of the sequences were trimmed with Trimmomatic v0.39 [Fish were caught and sacrificed by authorized persons immediately prior to excision of acanthocephalans from the guts. As for the processing of the eoacanthocephalans vis s.l. . We analic v0.39 . For moric v0.39 . While wP. laevis transcriptome published recently under NCBI GenBank accession number GIBA00000000.1 [https://sourceforge.net/projects/bbmap/) to the reference. Since 92–96% of reads mapped successfully under default settings, the transcriptome seems to be quite complete and serves as a useful resource for the analysis.As reference we used the 000000.1 . This tr000000.1  from mal000000.1  against Transcript quantification was done with the RSEM v1.3.3  softwarehttps://www.gbif.org/tool/81287/r-a-language-and-environment-for-statistical-computing) [Transcript abundance analyses were carried out with the Bioconductor package DESeq2 v1.28.1  in R v4.mputing) . Since wp-value ≤0.05, each). Corresponding genes were regarded to be expressed independently of sex and host, suggestive of their constitutive expression.As an approximation of targetability, we only kept genes having at least 50 transcript reads in each of the 20 samples. Then, we extracted all significantly unregulated genes, according to likewise transcript read counts in all four DESeq2 pairs of comparisons delineated above. In detail, retention of a gene required a maximum log fold change of 1.50 between at least two of the four groups of comparison  (see Supplementary Notes SOpen reading frames (ORFs) were extracted from candidate target transcripts running getorf within EMBOSS v6.5.7  with defsensulato): Adineta vaga (GCA_000513175.1), Adineta ricciae (GCA_900240375.1), Brachionus calyciflorus (GCA_002922825.1), Brachionus koreanus (GCA_009177125.1), Brachionus plicatilis (GCA_010279815.1), and Seison nebaliae (PRJEB43415). All sequences yielding hits with E-values ≤1e-50 were regarded evolutionarily conserved between Acanthocephala and the taxa compared. Corresponding hits were excluded from further analyses.Exclusion of sequences with similar sequences in non-acanthocephalan taxa was accomplished by blastp searches against the SwissProt database. For the same reason, we carried out tblastn searches against the genomes of six species from Bdelloidea, Monogononta, and Seisonidea, i.e., the three other higher-ranked taxa within the Rotifera-Acanthocephala clade commonly referred to as Syndermata or just Rotifera , operated in data-dependent acquisition mode with a top20 method. During the 120 min measurement, peptides were eluted with an optimized 5–40% acetonitrile/water gradient. Raw files were processed with MaxQuant v1.6.5.0 , 108 usiAbundances of matched proteins (iBAQ) and transcripts (transcript read counts) was carried out with Excel 2019 (Microsoft).https://www.genome.jp/tools/motif/; accessed 2021-09-01) and for PROSITE protein domains and functional sites [https://prosite.expasy.org/; accessed 2021-09-15). Additionally, potential enzyme functions were predicted by ECPred [Sequences of candidate target proteins were screened for PFAM protein motifs and domain features  at Kyotoal sites  at the Sy ECPred .https://www.bioinformatics.org/sms2/protein_gravy.html). N-terminal pre-sequences were predicted by TargetP v2.0 [https://biolib.com/DTU/DeepTMHMM). Prediction of subcellular localization based on sequence information was accomplished by the deep learning algorithm DeepLoc v1.0 [Protein’s grand average of hydropathy  was calcetP v2.0  and tranLoc v1.0 .https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb) [Protein 3D structures were modeled with AlphaFold2  as execu2.ipynb) . We appl2.ipynb)  for sequ2.ipynb)  applying2.ipynb) , 118.Protein-ligand binding site prediction was carried out with COACH-D  on protehttps://clinicaltrials.gov/), DrugBank [Ligands predicted to bind with most favorable free energy to binding sites in the candidate target proteins were further evaluated in the online databases ChEMBL , ClinicaDrugBank , and PubDrugBank . SpecialDrugBank .Additional file 1: Supplementary Note S1. Assembly of Neoechinorhynchus buttnerae draft genome. Supplementary Note S2. Assembly of Neoechinorhynchus agilis draft genome. Supplementary Table S1. Transcript abundance differences of candidates. Supplementary Table S2. Amino acid composition of candidate target proteins. Supplementary Table S3. Properties of target proteins. Supplementary Table S4. PFAM motifs of target proteins. Supplementary Table S5. 3D structure prediction of target proteins. Supplementary Table S6. Virtual ligand screening results. Supplementary Table S7. Bacterial genome assemblies used for data decontamination. Supplementary Table S8. Annotation of the mitochondrial genome of N. buttnerae. Supplementary Table S9. Specimens used for mass spectrometry. Supplementary Table S10. Standard InChI keys and 2D structures of candidate ligands. Supplementary Figure S1. Target identification workflow at Galaxy.Additional file 2: Supplementary Tables S3-S6.


Schistosoma, is a neglected disease with high global prevalence, affecting more than 240 million people in several countries. Praziquantel (PZQ) is the only drug currently available for the treatment. S. mansoni NTPDases  are potential drug targets for the discovery of new antischistosomal drugs. In this study, we screen NTPDases inhibitors from Centella erecta (Apiaceae) using an ultrafiltration combined UHPLC-QTOF-MS method and potato apyrase, identifying asiaticoside as one of the apyrase-binding compounds. After isolation of asiaticoside from C. erecta extract, we assessed its in vivo antischistosomal activities against Schistosoma mansoni worms and its in vitro enzymatic apyrase inhibition. Also, molecular docking analysis of asiaticoside against potato apyrase, S. mansoni NTPDases 1 and 2 were performed. Asiaticoside showed a significant in vitro apyrase inhibition and molecular docking studies corroborate with its possible actions in potato apyrase and S. mansoni NTPDases. In mice harboring a patent S. mansoni infection, a single oral dose of asiaticoside (400 mg/kg. p.o.) showed significantly in vivo antischistosomal efficacy, markedly decreasing the total worm load and egg burden, giving support for further exploration of apyrase inhibitors as antischistosomal agents.Schistosomiasis, caused by parasites of the genus  Schistosoma, is a neglected tropical disease with an estimated global prevalence of 240 million infected people in 78 countries and significant mortality and social and economic consequences − adduct ion in the negative mode with m/z 993.4827. The other fragment ions were at m/z 487.3400 [M-H-2Glc-Rha]− and at m/z 469.1537 [M-H-2Glc-Rha-H2O]−, which are in accordance with MS/MS studies and fragmentation pattern previously reports to asiaticoside − found; 993.4831 [M + Cl]− calculated. Purity of the isolated asiaticoside was estimated to be higher than 95% by 1H NMR data analysis.As asiaticoside  was identerature : 1H NMR 50 value, asiaticoside inhibited potato apyrase activity by approximately 43% at 100 µM, while at 50 µM potato apyrase activity was inhibited by about 33%  against potato apyrase was investigated. Asiaticoside was able to decrease the enzymatic activity of potato apyrase . Althougbout 33% .To investigate possible modes of asiaticoside interaction with target enzymes at the active sites, we performed molecular docking simulations. As shown in p < 0.001) in comparison with control group (p < 0.001) (p < 0.001) after treatment with a single oral dose of asiaticoside (400 mg/kg) in comparison with control group (p < 0.001) and 93.6% (p < 0.001) after the oral treatment with asiaticoside and PZQ, respectively (p < 0.001) and 94.4% (p < 0.001) after the oral treatment with asiaticoside and PZQ, respectively  showed a significant total worm burden reduction of 65.4% (ol group . PZQ (40< 0.001) . In addiol group . In feceectively A. Additiectively B.S. mansoni [S. mansoni, also being effective in inhibiting both potato apyrase and SmNTPDases [C. erecta showed as a potential source of apyrase inhibitors (data not shown). Based on these findings and corroborating with the high homology degree between SmNTPDases and potato apyrase [C. erecta extract.The discovery of new antischistosomal compounds is crucial in terms of public health, since schistosomiasis remains as the second most important human parasitic disease ,4,41. Du mansoni . AdditioNTPDases . In addi apyrase , we usedC. erecta extract were carried out for identification of apyrase ligands. The particular ligands from C. erecta extract may bound to the apyrase sites, forming enzyme-ligand complexes during the incubation process, which, due to their macromolecular size, would remain in the ultrafiltration membrane when centrifuged, while the unbound small compounds could pass through the membrane [C. erecta were subjected to UHPLC-ESI-QTOF-MS identification. According to UHPLC-ESI-QTOF-MS analysis, the ultrafiltration process produced an ultrafiltrate enriched mainly with three compounds (peaks 1–3) with binding affinity for apyrase, although a little difference between the LCMS chromatograms of the crude C. erecta extract and the ultrafiltrate was observed. These main compounds in ultrafiltrates were analyzed by their mass spectrometry data, accurate molecular weight, and fragment ions and annotated as asiaticoside (peak 1), madecassic acid (peak 2), and asiatic acid (peak 3). Although other possible ligands might be presented in the C. erecta extract (such as in Rt= 9.61 min), only three target compounds were annotated as potential apyrase inhibitors. Among them, asiaticoside was the only successfully isolated from C. erecta extract by chromatographic fractionation and further chemically identified by 13C- and 1H- NMR data analysis. Centella species consist mainly of triterpenoid saponins, in which asiaticoside is one of most studied compounds [Typically, an affinity ultrafiltration LC-MS screening involves three steps: incubation, screening by ultrafiltration and characterization by MS . It is kmembrane . After, ompounds .However, binding of a ligand target compound to the enzyme does not mean that it is an enzyme inhibitor, due to the possibility of non-specific binding of the compound to non-functional sites on the enzyme . Then, iAdditionally, to investigate how asiaticoside might bind to the active sites of NTPDases enzymes, asiaticoside was docked to active pockets of potato apyrase and SmNTPDases 1 and 2. Our results indicate that asiaticoside is able to interact with important catalytic residues of apyrase and SmNTPDases, providing a possible inhibition of these target proteins. In addition, we also observed that asiaticoside preferably interacted in the positive electrostatic region of the catalytic site from target proteins, similar that results obtained to cardamonin .S. mansoni mouse model. The in vivo treatment with asiaticoside was performed in mice harboring patent S. mansoni infection. To our knowledge, asiaticoside has not been yet evaluated against Schistosoma sp. After oral treatment at a single dose (400 mg/kg) of asiaticoside, worm burden was significantly reduced in comparison with the control group. Additionally, when compared to the reference drug PZQ (400 mg/kg), which reduced the parasitic burden in ~90% [Centella extracts (with high concentration of asiaticoside) did not show any adverse effects by oral administration in mice at least up to 1 g/kg [C. asiatica extract (containing ~46% of asiaticoside) exhibited LD50 > 2 g/kg by oral administration in rats [Then, considering the importance of apyrase inhibitors as potential antischistosomal compounds and keeping in view the above-mentioned experimental approach, we evaluate the in vivo activity of asiaticoside in the  in ~90% , asiatico 1 g/kg ,45. Furt in rats .Actinopyga echinites and Holothuria polii have interesting in vitro schistosomicidal activity [Vernonia amygdalina (Asteraceae) were in vitro and in vivo evaluated against S. japonicum [Pulsatilla chinensis (Bunge) Regel, exhibits antischistosomal properties in vivo against S. japonicum and S. mansoni [Moreover, there are few reports in the literature regarding the schistosomicidal activity of saponins. Previous studies showed that saponins from the marine organisms activity . Also, iaponicum , showing mansoni .S. mansoni to the organism are due to eggs in the host’s tissues, triggering inflammatory reactions and granuloma formation [S. mansoni. After the treatment with asiaticoside the number of eggs per gram (OPG) was also markedly reduced in feces examined by the Kato–Katz method. Moreover, the oogram pattern showed that the oral administration of asiaticoside produced significant reductions in immature and mature eggs. Taken together, our results on egg burden agree with the noticeable ability of asiaticoside in reducing adult worms of Schistosoma.In addition, it is known that the main lesions caused by ormation ,52. Thenormation , the theSchistosoma tegument is the interface between the environment and the parasite, achieving vital functions for the worms, such as nutrient absorption, excretion, protection against the host’s immune system [Schistosoma tegument are some essential enzymes, such as the SmNTPDases, which are important for the survival of Schistosoma worms inside the host [The e system . In Schithe host ,31,53. Tthe host , other mthe host  should bC. erecta extract. Madecassic and asiatic acids were also annotated as apyrase-binding compounds by this method. Asiaticoside was isolated from C. erecta extract and further in vitro assays confirmed its potato apyrase inhibitory activity. Additionally, molecular modeling and docking assays were used to evaluate the interaction of asiaticoside with the active sites of SmNTPDase 1, SmNTPDase 2, and potato apyrase. In addition, our findings demonstrated, for the first time, that asiaticoside has markedly in vivo antischistosomal efficacy in mice harboring a patent S. mansoni infection, markedly decreasing the total worm load and egg burden. However, more clinical studies are needed to validate the use of asiaticoside as a therapeutic schistosomicidal agent. Additionally, future studies are also in progress to evaluate the antischistosomal properties of madecassic and asiatic acids.In this study, an ultrafiltration combined with UHPLC-QTOF-MS approach was used to identify the asiaticoside as a potential apyrase inhibitor from


Aerosolized glucocorticoid treatment was independently associated with bacterial isolation in these patients. Acinetobacter baumannii is a nosocomial pathogen associated with severe illness and death. Glucocorticoid aerosol is a common inhalation therapy in patients receiving invasive mechanical ventilation. We conducted a prospective cohort study to analyze the association between glucocorticoid aerosol therapy and A. baumannii isolation from ventilator patients in China. Of 497 enrolled patients, 262 (52.7%) received glucocorticoid aerosol, and A. baumannii was isolated from 159 (32.0%). Glucocorticoid aerosol therapy was an independent risk factor for A. baumannii isolation . Patients receiving glucocorticoid aerosol had a higher cumulative hazard for A. baumannii isolation and analysis showed that glucocorticoid aerosol therapy increased A. baumannii isolation in most subpopulations. Glucocorticoid aerosol was not a direct risk factor for 30-day mortality, but A. baumannii isolation was independently associated with 30-day mortality in ventilator patients. Physicians should consider potential A. baumannii infection when prescribing glucocorticoid aerosol therapy. Acinetobacter baumannii, a gram-negative coccobacillus, is a major nosocomial pathogen worldwide. A. baumannii is particularly challenging in intensive care units (ICUs). According to the Extended Prevalence of Infection in Intensive Care study, aimed at providing information on the prevalence of infection in ICUs worldwide, Acinetobacter spp. constituted 8.8% of all culture-positive ICU infections in 2007 . We used Escherichia coli (ATCC accession no. 25922) and Pseudomonas aeruginosa (ATCC accession no. 27853) as quality controls.We performed microbial cultures according to standard procedures. In brief, we incubated respiratory samples on MacConkey agar plates at 5% CO>48 hours after enrollment and before A. baumannii isolation, with or without nonglucocorticoid aerosolized medications for any duration. We defined aerosol inhalation without glucocorticoid as patients who received only aerosolized nonglucocorticoid medications, such as bronchodilators and expectorants, for >48 hours after enrollment and before A. baumannii isolation. We excluded all other conditions.We defined no aerosol inhalation as patients who did not receive aerosolized medications during the study. We defined glucocorticoid aerosol therapy as patients who received aerosolized glucocorticoids for A. baumannii isolation, which we defined as A. baumannii–positive culture from the lower respiratory tract samples collected during the ICU stay. Negative outcomes were no A. baumannii isolation before death, ICU discharge, or end of follow-up period. We recorded the time-to-event, which we defined as number of days from enrollment to A. baumannii isolation.The primary endpoint was A. baumannii risk factors, such as use of broad-spectrum antimicrobial drugs, invasive mechanical ventilation, urethral catheter placement, vasopressor treatment, renal dialysis, and length of ICU stay. In addition, we recorded indications for glucocorticoid aerosol therapy by reviewing patients’ medical records. We reviewed patients’ clinical data to determine A. baumannii isolation status as infection, colonization, or undefined.We collected baseline information at ICU admission, including age, sex, history of smoking and surgeries, underlying conditions, past inhaled steroids for chronic conditions, and Charlson comorbidity index. We used the Acute Physiology and Chronic Health Evaluation II (APACHE II) score to assess illness severity. We also recorded other possible A. baumannii isolation and 30-day mortality. We performed propensity score matching analysis to reduce the imbalance between the glucocorticoid aerosol therapy and nonglucocorticoid groups. We included all possible covariables  in the propensity score matching. We calculated propensity scores by using a logistic regression model. We applied a 1:1 nearest neighbor matching algorithm with a caliper of 0.02 and without replacement. We assessed balance of variables in both groups by standardized differences. We analyzed A. baumannii isolation in complete cases and the propensity-matched cohort. We used Kaplan-Meier curves to visually compare cumulative hazards for A. baumannii isolation among the 3 groups, which we evaluated by using a log-rank test. To assess the consistency of glucocorticoid therapy in terms of its effect on A. baumannii isolation from prespecified subgroups with different characteristics, we applied Cox proportional hazards model with Efron’s method for handling ties and used forest plots for HRs and 95% CIs. We assessed heterogeneity of efficacy of glucocorticoid therapy on A. baumannii isolation in subgroups by using an interaction test, expressed p values for interaction, and considered p<0.05 statistically significant. We performed all analyses by using SPSS Statistics 16.0  and R version 3.0 .We expressed continuous variables as median and interquartile range (IQR) or mean and SD and categorical variables as number and percentage. We used univariate and multivariate Cox proportional hazards regression and hazard ratio (HR) and 95% CI to assess risk factors for A. baumannii from 159 (32.0%) patients. The median patient age was 60.1 (IQR 49–73) years, and 67.8% were male. The median length of ICU stay was 15 (IQR 7–23) days. Besides A. baumannii, the 3 other bacteria commonly isolated were Klebsiella pneumoniae , P. aeruginosa , and E. coli   received no aerosol inhalation, 262 (52.7%) received glucocorticoid aerosol, and 98 (19.7%) received aerosol inhalation without glucocorticoid . We isol5, 3.0%) Table 1.5, 3.0%) Table 2.A. baumannii isolation . Aerosol inhalation without glucocorticoid was not a risk factor for A. baumannii (p>0.05). Other candidate risk factors were cardiovascular diseases, chronic renal insufficiency, COPD or asthma, current or former smoking history, use of broad-spectrum antimicrobial drugs for >7 days, invasive mechanical ventilation for >5 days, vasopressor treatment, renal dialysis for >3 days, and APACHE II score.We performed univariate Cox regression analysis of risk factors for solation . CompareA. baumannii isolation, we established 2 models using multivariate Cox regression analysis in complete cases. Model 1 included all variables, and model 2 only included variables with p<0.1 in the univariate analysis. Glucocorticoid aerosol was an independent risk factor for A. baumannii isolation in both model 1  (A. baumannii (A. baumannii isolation (To assess whether glucocorticoid aerosol therapy was an independent risk factor for = 0.049)  Table 3 = 0.049) . Cardiovaumannii  Table 3.solation  Table 4.A. baumannii isolation  and aerosol inhalation without glucocorticoid  groups . The cumulative hazard for ) groups .A. baumannii isolation from different subpopulations. We found glucocorticoid aerosol was a promoting factor for A. baumannii isolation from most subpopulations, except patients with type 2 diabetes mellitus, hematologic malignancy, antimicrobial drug use for A. baumannii, and short ICU stays (p>0.05) (A. baumannii isolation was relatively greater in the subgroup of patients with longer vasopressor treatment (interaction p = 0.006)  . We note= 0.006) .A. baumannii isolation was independently associated with 30-day mortality   (We performed univariate and multivariate Cox regression analyses to evaluate the possible risk factors for 30-day mortality in critically ill patients on ventilators. We found glucocorticoid aerosol was not a risk factor for 30-day mortality in those patients, but A. baumannii–positive cultures from lower respiratory tract samples in 3 ICUs in China. After controlling for other variables, our findings showed that glucocorticoid aerosol increased the risk for A. baumannii isolation from critically ill patients on invasive mechanical ventilation.In this study, we assessed the effect of the commonly used glucocorticoid aerosol therapy on the frequency of A. baumannii is ubiquitous in nature and is becoming more frequent in hospitals. In our study, 32% of patients acquired A. baumannii during the 30-day follow-up period. Over the past 2 decades, several studies have attempted to characterize and identify risk factors for A. baumannii colonization or infection. Invasive operations, such as endotracheal mechanical ventilation, inserted invasive devices, ICU stays, recent surgery, use of broad-spectrum antimicrobial drugs, ineffective antimicrobial therapy, and septic shock at diagnosis, are risk factors for MDR A. baumannii colonization or infection and for death (A. baumannii isolation from ventilated patients, which is consistent with previous studies (A. baumannii bloodstream infection (A. baumannii isolation. Another population-based study reported that patients with chronic heart failure had a markedly increased risk for hospitalization with pneumonia (A. baumannii isolation remain unknown.We determined that prolonged use of broad-spectrum antimicrobial drugs, invasive mechanical ventilation, and vasopressor treatment were independent risk factors for In critically ill patients undergoing mechanical ventilation, aerosol inhalation is a common intervention for treating various pulmonary diseases. An international survey demonstrated that 99% of 611 ICUs from 70 countries reported using aerosol therapy during mechanical ventilation, including noninvasive ventilation, and the most frequently delivered drugs were bronchodilators and steroids (A. baumanni–related pneumonia is associated with severe illness and death, we chose A. baumannii isolation as an outcome and explored its relationship with glucocorticoid aerosol therapy. Our study showed that glucocorticoid aerosol therapy was an independent risk factor for A. baumannii isolation from patients on ventilators. Compared with no aerosol inhalation, glucocorticoid aerosol inhalation increased the risk for A. baumannii by ≈1.5 times. Although further analysis revealed that glucocorticoid aerosol was not directly associated with 30-day mortality, it still might contribute to poor clinical prognosis due to its effect on A. baumannii isolation. As we described, A. baumannii, especially MDR A. baumannii pneumonia, is well recognized as a risk factor for death. In this study, we also found A. baumannii isolation was an independent risk factor for 30-day mortality in patients receiving invasive mechanical ventilation. Because glucocorticoid aerosol heightened the likelihood of acquiring A. baumannii, it might exert a secondary effect, death among A. baumannii–infected patients. Thus, further investigation in a much larger patient population could describe a downstream mortality effect of glucocorticoid aerosol therapy. Ventilated patients are already vulnerable to pneumonia. Therefore, evaluating whether commonly used aerosol therapy increases the risk for nosocomial pneumonia is crucial, especially when inhaled with glucocorticoids. Because A. baumannii isolation. Because these structural or underlying lung diseases and severe acute lung injury necessitate longer duration of mechanical ventilation, our results were compatible with previously described risk factors for A. baumannii infection. In contrast to glucocorticoid aerosol, we did not detect an association between aerosol inhalation without glucocorticoid and A. baumannii. Because both therapies generate aerosols, our previous concern that aerosols were a source of A. baumannii acquisition might not be reasonable.When we included glucocorticoid aerosol indications in multivariate analysis, we found COPD and asthma and possible ARDS were independent risk factors for A. baumannii isolation remain elusive. Previously considered sterile, healthy lungs harbor complex and dynamic microbiota communities (Streptococcus pneumonia infection (Streptococcus (Prevotella bacteria in concert with downregulation of genes promoting epithelial defense associated with inhaled corticosteroid use (A. baumannii isolation from patients on ventilators.Reasons why glucocorticoids increase the risk for A. baumannii isolation when receiving glucocorticoid aerosol. Based on our findings, we recommend that intensive care teams more carefully consider the risk of widespread usage of glucocorticoid aerosol in patients with invasive mechanical ventilation. Because glucocorticoid aerosol therapy had a much greater favorable eﬀect on A. baumannii isolation in the subgroup of patients on vasopressors for >3 days, clinicians should be particularly cautious about giving glucocorticoid aerosol to patients on long vasopressor treatments. Because patients with diabetes, hematologic malignancy, and shorter ICU stays were at relatively lower risk for glucocorticoid aerosol–associated A. baumannii acquisition, glucocorticoid aerosol could be considered when appropriate for indications in these patients. Altogether, our study suggests ICU teams need to identify the specific patient subgroups that will truly benefit from glucocorticoid aerosol therapy rather than more generalized administration. Limiting glucocorticoid aerosol use might be considered as part of existing antimicrobial stewardship bundles. In addition, defining the duration of glucocorticoid aerosol therapy might help maximize benefit while reducing associated risk. Interventional studies exploring the effects of different glucocorticoid aerosol therapy durations on the occurrence of various types of nosocomial pneumonia are needed. Clinicians should individualize patient care and manage treatments on the basis of subgroup analysis results. Our results showed that, in most subpopulations, regardless of the presence or absence of the prespecified characteristics, patients were at higher risk for A. baumannii isolation remain, even after adjusting by subgroup analysis, propensity score matching analysis, and multivariable Cox regression. A similar situation exists for the analysis of risk factors for death. Second, a potential time bias remains, in which patients might have been exposed to risk factors before study enrollment. However, we have excluded patients with prolonged exposure to several well-known risk factors to minimize the possible effect of time bias. Third, exclusion of certain cases might pose a potential selection bias, including survivorship bias. Future randomized controlled interventional studies are expected to confirm our findings and minimize the effects of confounders and the above biases. Fourth, the study included only 3 ICUs in China and did not focus on pathogens other than A. baumannii. Research involving more centers and more cases could explore whether glucocorticoid aerosol is a common risk factor in the overall nosocomial pneumonia risk.The first limitation of our study is that, because it was an observational study, confounders that might influence the effect of glucocorticoid aerosol on A. baumannii isolation from patients receiving invasive mechanical ventilation. Because of high mortality rates associated with A. baumannii–related nosocomial pneumonia, clinicians should carefully consider both the beneficial and harmful effects of glucocorticoid aerosol before administering this therapy.In conclusion, we found glucocorticoid aerosol therapy was an independent risk factor for Acinetobacter baumannii among patients receiving glucocorticoid aerosol therapy during invasive mechanical ventilation, China.Additional information on


R2 = 0.93, Q2 = 0.53), with evidence of dysregulated tryptophan and purine metabolism. Nine urine metabolites differed significantly in animals with acute kidney injury with a pattern suggestive of increased aerobic glycolysis. Dysregulated metabolites in kidney tissue and urine did not overlap. CPB/DHCA strongly affected the serum metabolic profile, with only one metabolite that differed significantly with acute kidney injury . In conclusion, based on these findings, kidney tryptophan and purine metabolism are candidates for further mechanistic and therapeutic investigation. Urine biomarkers of aerobic glycolysis could help diagnose early acute kidney injury after CPB/DHCA and warrant further evaluation. The serum metabolites measured at this early time point did not strongly differentiate based on acute kidney injury.Acute kidney injury (AKI) is a common cause of morbidity after congenital heart disease surgery. Progress on diagnosis and therapy remains limited, however, in part due to poor mechanistic understanding and a lack of relevant translational models. Metabolomic approaches could help identify novel mechanisms of injury and potential therapeutic targets. In the present study, we used a piglet model of cardiopulmonary bypass with deep hypothermic circulatory arrest (CPB/DHCA) and targeted metabolic profiling of kidney tissue, urine, and serum to evaluate metabolic changes specific to animals with histological acute kidney injury. CPB/DHCA animals with acute kidney injury were compared with those without acute kidney injury and mechanically ventilated controls. Acute kidney injury occurred in 10 of 20 CPB/DHCA animals 4 h after CPB/DHCA and 0 of 7 control animals. Injured kidneys showed a distinct tissue metabolic profile compared with uninjured kidneys (NEW & NOTEWORTHY This project explored the metabolic underpinnings of postoperative acute kidney injury (AKI) following pediatric cardiac surgery in a translationally relevant large animal model of cardiopulmonary bypass with deep hypothermic circulatory arrest. Here, we present novel evidence for dysregulated tryptophan catabolism and purine catabolism in kidney tissue and increased urinary glycolysis intermediates in animals who developed histological AKI. These pathways represent potential diagnostic and therapeutic targets for postoperative AKI in this high-risk population. Acute kidney injury (AKI) is common following congenital heart disease (CHD) surgery with an incidence of >25% –5. PostoMetabolomics represents a novel approach to the study of AKI , 19. MetOur group and others have begun to characterize the metabolomic changes caused by CHD surgery –27. CHD Animal models are particularly useful for studying metabolomics as metabolic pathways are highly conserved across species, in contrast to protein biomarkers that demonstrate significant heterogeneity. Several studies have sought to identify changes in kidney tissue metabolomic profiles in small and large animal models of ischemia-reperfusion using vascular clamping strategies –34. HoweIn this study, we used our translationally relevant large animal model of pediatric CPB/DHCA in conjunction with targeted metabolic profiling by liquid chromatography-tandem mass spectroscopy (LC-MS/MS) to determine the kidney tissue, urine, and serum metabolic profiles in infant piglets exposed to CPB/DHCA and anesthesia-only controls. Metabolic profiling studies are typically the first stage of metabolomic research , used toThe animals used in this study were part of a parent study on the effects of bovine intestinal alkaline phosphatase infusion on AKI during CPB/DHCA . Kidney Immediately before euthanasia, whole blood for serum metabolomic profiling was obtained from the femoral arterial line and urine was obtained by direct bladder puncture. Urine and serum samples were frozen at −80°C for batch analysis. Following euthanasia, the right kidney was harvested, and the tissue was flash-frozen in liquid nitrogen for metabolic profiling or placed in 10% formalin for histology. Serum creatinine, serum and urine neutrophil gelatinase-associated lipocalin (NGAL), and kidney NGAL and kidney injury molecule-1 (KIM-1) mRNA were measured as previously published .Kidney tissue fixed overnight in 10% formalin was then paraffin-embedded and sectioned at 4 µm. Sections were oriented to demonstrate a radial section of the cortex and medulla. Serial sections were stained with hematoxylin and eosin and evaluated for the severity of histological kidney injury  by a blinded board-certified pediatric pathologist with a specialization in pediatric kidney disease (A.T.) using a previously published methodology , 38–41. g for 20 min at 4°C. The precipitant was reextracted with an additional 500 µL of ice-cold methanol-water [80/20 (vol/vol)], and the supernatants were combined, centrifuged, and dried in a SpeedVac concentrator . Serum and urine samples (100 µL) were mixed with 400 µL ice-cold methanol, sonicated for 10 min, and incubated for 4 h at −80°C for protein precipitation. This was followed by centrifugation at 14,000 g for 20 min at 4°C and drying in a SpeedVac concentrator. All samples were reconstituted with 60 µL of water-methanol [80:20 (vol/vol)] and transferred into HPLC vials with inserts for analysis.Frozen tissue samples were extracted according to a previously published protocol . Frozen https://doi.org/10.6084/m9.figshare.19666416.v1). Yuan et al. in their study have demonstrated reproducibility and extraction recovery testing using this protocol in formalin-fixed paraffin-embedded normal kidney tissue as well as in kidneys of patients with acute myeloid leukemia. Our samples were immediately frozen and processed quickly using the previously published protocol; thus, no negative impact regarding metabolite stability or recovery was expected.Multiple reaction monitoring of 235 metabolites using a positive/negative ion-switching 5500 QTRAP HPLC-MS/MS was used for analysis as previously published , 43. Thesolvent B; and from 3 to 22 min, the percentage of solvent B was decreased from 85% to 2% and was kept at 2% for additional 3 min. At minute 26, solvent B was increased again back to 85% and the column flushed for an additional 7 min at 85% solvent B. Once the data were acquired, MultiQuant  software was used for data processing of 235 unique metabolites. For between-sample normalization, the intensity values for each sample were summed, and the median value of the sums across all samples were determined. Tune and quality control samples were evenly distributed during the batches. The intensity values of each sample were then scaled such that the sum of the scaled intensities equaled the median value of all samples. Urinary metabolites were also normalized to urine creatinine. MS analysis was performed by a blinded investigator (J.K.).Sample analysis was performed using an Agilent 1200 series HPLC system  interfaced with an ABSciex 5500 hybrid triple quadrupole/linear ion trap mass spectrometer  equipped with an electrospray ionization source operating in the positive/negative switch mode. The Q1 (precursor ion) and Q3 (fragment ion) transitions, the metabolite names, dwell times, and the appropriate collision energies for both positive and negative ion modes were adapted from a study by Yuan et al. with several metabolite transitions added by our group . In total, Q1 and Q3 transitions were set to unit resolution for optimal metabolite ion isolation and selectivity. In addition, the polarity switching (settling) time was set to 50 ms. In 1.42 s using a 3-ms dwell time, we were able to obtain 6–14 scans per metabolite peak. Eight microliters of sample were injected onto an Amide XBridge HPLC column . The mobile phases consisted of “HPLC buffer A”  and “HPLC buffer B” (100% acetonitrile). The HPLC settings were as follows: from 0 to 3 min, the mobile phase was kept at 85% n = 27, 20 CPB/DHCA animals and 7 controls). Our primary outcome was differences in the metabolic profile and individual metabolites of kidney tissue from the following three groups: mechanically ventilated controls, CPB/DHCA animals without AKI (AKI score = 0), and CPB/DHCA animals with AKI (AKI score = 1–4). Secondary outcomes were the difference in the metabolic profile and individual metabolites in serum and urine from the three groups and the comparison of the metabolic profiles of kidney tissue, serum, and urine. Metabolite relative peak intensities  were analyzed using MetaboAnalyst 5.0   . All anilyst.ca) . PartialA total of 27 animals were studied . No control animals developed histological AKI. Of the CPB/DHCA animals, 50% developed histological AKI with proximal tubular injury  in >5% of tubules on low-powered microscopic examination as previously published . DetailsR2 = 0.99, Q2 = 0.99). At an individual metabolite level, 98% (231 of 235 metabolites) of the individual metabolites tested differed by sample type at a false discovery rate of <0.05. No difference in the metabolic profile was seen based on exposure to bovine intestinal alkaline phosphatase . The complete metabolite data set is provided in Supplemental Data S2 (https://doi.org/10.6084/m9.figshare.19181945.v1).In our metabolomic analysis, we first examined the differences in the tissue, circulating (serum), and urinary metabolic profiles across all 27 animals to assess how closely urinary and circulating metabolites mirrored the relative quantities of metabolites in kidney tissue. Samples clustered very tightly by source (tissue vs. serum vs. urine) on both unsupervised (PCA) and supervised (PLS-DA) dimension reduction analysis A, indicaR2 = 0.93, Q2 = 0.53; Next, we evaluated how the kidney tissue metabolic profile and individual metabolites varied across the following three groups: CPB/DHCA animals with AKI, CPB/DHCA animals without AKI, and mechanically ventilated controls (no AKI). The three groups were readily distinguished by their metabolic profile with moderate reproducibility on cross-validation , with the most notable separation occurring between mechanical ventilation controls and CPB/DHCA animals  is the primary route of tryptophan catabolism. It produces multiple biologically active intermediates, and its end product, quinolate, is the substrate for de novo nicotinamide adenine dinucleotide (NAD) production. During pathological inflammatory states, flux through the KP is increased due to the upregulation of the first pathway enzyme  across multiple organs –48. EvidN-methyl-d-aspartate receptor antagonist properties  also reduces AKI in both ischemia-reperfusion and cisplatin models via an increase in de novo NAD production  in noncardiac surgery-associated AKI. Administration of exogenous kynurenic acid decreases ischemia-reperfusion-induced AKI in mice , and blooperties . Reductioduction , whereasoduction .For the first time, we now have evidence in both pediatric patients and our complex large animal model of infant CPB/DHCA that the KP is also activated in cardiac surgery-induced AKI. Infant cardiac surgery results in a significant increase in proximal KP metabolites that peaks shortly after surgery and returns to baseline in most cases by 48 h postoperatively . CirculaOur second prominent finding in injured kidney tissue was an increase in the end products of purine catabolism, particularly xanthosine, uric acid, and allantoin, which has not previously been identified in cardiac surgery-induced AKI. Uric acid is produced primarily in the liver and excreted through the kidney after sequential reabsorption and secretion in the proximal tubule . Uric acUnlike our findings in kidney tissue, the urinary metabolites that most strongly differed by exposure to CPB/DHCA and AKI were concentrated within glycolysis and the proximal TCA cycle. These metabolites were typically elevated in animals exposed to CPB/DHCA with further elevation in animals demonstrating histological AKI. Both lactate and ribose-5-phosphate (pentose phosphate pathway) were also increased despite adequate oxygen delivery postbypass and no evidence of impaired flow into the TCA cycle, consistent with increased glycolytic flux and the Warburg effect (aerobic glycolysis). To our knowledge, the Warburg effect has not previously been identified in cardiac surgery-induced AKI. The Warburg effect has been well described in oncology, however, and there is increasing evidence that it may also occur in less complex small animal models of noncardiac surgery-induced AKI. Septic and ischemia-reperfusion models of AKI result in the upregulation of glycolysis and decreased gluconeogenesis , 62. FurIt is not clear from our study why glycolysis intermediates were significantly altered in urine but not in the kidney tissue itself. This discrepancy could be related to differences between intracellular metabolite concentrations and the effects of altered proximal tubular function on urinary metabolite concentrations. In addition, changes in tissue metabolites could reflect more severely injured, nonfunctional nephrons that do not contribute substantially to urine production. Even though changes in urine metabolites do not directly reflect tissue metabolite changes in this model, our findings suggest that urine biomarkers of increased glycolysis could be promising candidates for early AKI diagnosis after pediatric cardiac surgery. Furthermore, modulation of glycolysis should be further explored in translational models of cardiac surgery for the prevention of AKI.Serum metabolites were the least associated with AKI in this study, with only a single metabolite  that was significantly different in animals with histological AKI. Instead, differences in serum metabolic profiles across groups could largely be attributed to exposure to CPB/DHCA at this early time point. This finding is not surprising. We have previously demonstrated that infant cardiac surgery with CPB results in a profound shift in the circulating metabolome that continues to diverge from baseline for at least 24 h . In our Collectively, our findings highlight the opportunity created by using a large animal translational model to study kidney metabolomics and AKI after cardiac surgery. Most of our current understanding of kidney metabolic changes in AKI comes from either nonbypass small animal models  , 65, 66 Our study has several important limitations. First, the sample size is small compared with human metabolomic studies, limiting the power to detect true differences in metabolites with smaller fold changes and/or greater variability. The consistent finding of greater fold changes in metabolites from injured kidneys even when not statistically different suggests the presence of type 2 error. Second, kidney injury in this model is heterogeneous, and sampling variability itself could lead to misclassification. Future studies should be scaled to both increase the number of animals assessed and to allow for the comparison of multiple samples from both kidneys. Also, cell-specific metabolism changes should be defined using microdissection techniques. Third, this study focused on early kidney injury (4 h post-CPB) to understand the initial metabolic changes at a point where intervention might be more impactful. We did not define the natural history of metabolic changes following CPB/DHCA. Longitudinal studies with repeat urine/serum sampling, continuous functional measurements of filtration, and different euthanasia time points are necessary to understand the progression of metabolic changes, including through the recovery period. Finally, to decrease variability, only female animals were used in this study. We recently demonstrated sex differences in metabolomic and cardiorenal outcomes in a murine model of ischemic AKI . Future With our persistent failure to decipher postoperative organ injury after cardiac surgery, the scientific community is increasingly realizing that complex pathophysiology may require a matching complex research strategy to drive progress . In this10.6084/m9.figshare.19666416.v1https://doi.org/10.6084/m9.figshare.19666416.v1;Supplemental Data S1: 10.6084/m9.figshare.19181945.v1https://doi.org/10.6084/m9.figshare.19181945.v1.Supplemental Data S2: This work was supported by the Department of Defense Award PR152240 [to Principal Investigator (PI): J.A.D], American Heart Association Grant 17IRG33410724 (to PI: J.A.D.), and National Heart, Lung, and Blood Institute Grants K23HL123634 (to PI: J.A.D.) and R01HL156936 (to PI: J.A.D.).No conflicts of interest, financial or otherwise, are declared by the authors.J.A.D., J.J., S.L., D.E.S., S.O.-L., and J.K. conceived and designed research; J.A.D., J.R., L.K., S.O.-L., and J.K. performed experiments; J.A.D., L.K., A.T., and J.K. analyzed data; J.A.D., L.K., A.T., S.O.-L., and J.K. interpreted results of experiments; J.A.D. prepared figures; J.A.D. drafted manuscript; J.A.D., J.R., L.K., B.S.F., J.J., R.J.I., S.L., J.I., E.A., A.T., D.E.S., S.O.-L., and J.K. edited and revised manuscript; J.A.D., J.R., L.K., B.S.F., J.J., R.J.I., S.L., J.I., E.A., A.T., D.E.S., S.O.-L., and J.K. approved final version of manuscript.


Porphyromonas gingivalis is a primary causative agent of chronic periodontitis. Moreover, it leads to several systemic diseases, including rheumatoid arthritis, cardiovascular, neurodegenerative, and Alzheimer’s diseases. It seems that the development of a vaccine against this bacterium is necessary. Thus, this study decided to identify novel immunogenic targets and developed multiple epitope-based vaccines against P. gingivalis. For this purpose, the pan/core-proteome of this bacterium was studied, and the suitable vaccine targets were selected based on different properties, including exposed localization of proteins, antigenicity, non-allergenicity, non-similarity to host proteome, stability, B-cell epitopes and MHC II binding sites, sequence conservation, molecular docking, and immune simulation. Through the quartile scoring method, 12 proteins with ≥ 20 scores were considered as suitable immunogenic targets. The results of the protein domain and functional class search showed that most of the immunogenic proteins were involved in the transport and metabolism of inorganic ions and lipids. In addition, two unknown function proteins, including WP_004584259.1 and WP_099780539.1 were detected as immunogenic targets. Three constructions carrying multi-epitopes were generated including Naked, LCL, and as chimeric structures. Among them, FliC chimeric protein had the strongest affinity to the human TLR2, 4, and 6, while the LCL platform represented the highest level of immune stimulation response. The obtained results from this study revealed new insights into prophylactic routes against P. gingivalis by introducing novel immunogenic targets. However, further investigations, including site-directed mutation and immunoassay are needed to confirm the pathogenic role and protectivity of these novel proteins. Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia that colonize the oral cavity [P. gingivalis is a nonmotile, Gram-negative, obligately anaerobic, and rod-shaped bacterium that colonizes dental plaque biofilms in the human oral cavity, and is considered the primary causative agent responsible for the development of chronic periodontitis [Periodontitis is an inflammatory polymicrobial disease and one of the humans’ most common bacterial infections affecting nearly 50% of the global population . The patl cavity . P. gingdontitis . This ordontitis . It is cP. gingivalis has several virulence factors, including cysteine proteases (gingipains), heat shock proteins, lipopolysaccharide, major fimbriae, and capsule. The gingipains can degrade several host proteins, such as complement proteins, immunoglobulins, cytokines/chemokines, and host cell receptors [eceptors .P. gingivalis. However, in recent years, concerns have been raised about the efficacy of antimicrobials in treating infections associated with oral biofilm. Several studies have demonstrated P. gingivalis can survive antibiotic treatment and leads to the recurrence of chronic periodontitis [Several classes of antibiotics have been proposed to treat infections associated with dontitis , 7. The dontitis . VaccinaP. gingivalis by employing in silico approaches.With the advent of genome sequencing technology, a considerable revolution in immunization has occurred. Genomic databases have greatly facilitated the investigation of immunogenic candidates to develop new potential vaccine targets against pathogenic microorganisms . ReverseP. gingivalis strains with completely annotated genome sequences were retrieved from the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) and translated by CLC Genomics Workbench software . Pan/core-genome analysis was performed by the Bacterial Pan Genome Analysis tool (BPGA) [In this study, 17 l (BPGA)  the corehttps://www.psort.org/psortb/). Only outer membranes, extracellular, and secreted proteins were selected in this step [The subcellular localization of the proteins was identified using PSORTb version 3.0.3 (his step .http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html) with a cut-off value ≥ 0.5 [http://crdd.osdd.net/raghava/algpred/) with a cut-off ≥ 0.5 [The antigenicity of the putative immunogenic targets was predicted using the VaxiJen tool (ue ≥ 0.5 . Subsequff ≥ 0.5 .https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Protein) [All selected proteins were analyzed to determine sequence similarity to the human proteome  using the PSI-BLAST tool in the BLASTp database (Protein) . Sequenchttps://services.healthtech.dtu.dk/service.php?BepiPred-2.0) [http://tools.iedb.org/tepitool/) with a cut-off of the top 5% of peptides [Linear B-cell epitopes of all selected putative immunogenic proteins from the previous steps were identified with a threshold ≥ 0.6 using the BepiPred v2.0 tool (red-2.0) . The ratpeptides . The rathttps://webs.iiitd.edu.in/raghava/vicmpred/submission.html). The number of amino acids, molecular weight, theoretical pI value, estimated half-life, aliphatic index, and instability index were determined using the Expasy ProtParam server (https://web.expasy.org/protparam/) [http://www.Violinet.org/vaxign2). Adhesins are potential vaccine candidates due to their role in adherence, colonization, and bacterial survival [Physicochemical properties of proteins were analyzed using different databases. The functional class of the proteins was determined by the VICMpred database (tparam/) . The instparam/) . In addisurvival .The selected proteins were analyzed using the quartile method scoring using eight indicators, including functional class , antigenicity, hydropathy index, instability index, MHC-II binding site ratio, linear B-cell epitope ratio, conformational B-cell epitope, and adhesion probability value. The sum of all scores for each protein was considered the final score. Proteins with ≥ 20 points were considered suitable immunogenic targets .https://robetta.bakerlab.org/) [https://prosa.services.came.sbg.ac.at/prosa.ph). This server displays the potential errors in the 3D model [http://tools.iedb.org/ellipro/) was used to identify the conformational B-cell epitopes with a threshold value of ≥ 0.8. The predicted conformational B-cell epitopes were displayed on the surface of each protein in different colors using Jmol software .The tertiary structure (3D) of the putative immunogenic proteins was characterized using the Robetta tool (ab.org/) . The qua3D model . In addihttps://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml), and EggNOG (http://eggnog5.embl.de/#/app/home) were used to find the major protein domains. CDD is part of NCBI’s Entrez query and provides an annotation of protein sequences with the location of the conserved domain [Conserved Domain Database, CDD (d domain . EggNOG d domain .https://string-db.org/) to understand the interactions of putative vaccine candidates with unknown functions with other proteins of P. gingivalis to estimate their function. The connection scores > 0.5 were considered.In this part, we used the STRING database  [Linear B-cell epitopes were predicted using the BepiPred database (Threshold ≥ 0.6). The conformational epitopes are obtained using the ElliPro database (Threshold ≥ 0.8). The Conservancy of linear and conformational B-cell epitopes was determined among 17 genbank) . FinallyThree multiple epitope-based vaccines were generated using three different platforms including Naked, FliC, and LCL. Different arrangements of the selected epitopes with rigid (EAAAK) and flexible (GPGPG) linkers were developed, and the most antigenic models were chosen. The tertiary structures (3D) of the Naked, FliC, and LCL chimeras were modeled using the Robetta web-tool. The 3D structures were validated by the ProSA-web server and Ramachandran diagrams.https://life.bsc.es/pid/pydockweb/default/index) [https://kraken.iac.rm.cnr.it/C-IMMSIM/index.php) was used to predict immune simulations of multi-epitope-based vaccines.Molecular docking and the binding affinity of multi-epitope-based vaccines to human TLR-1 (PDB: 2Z7X), TLR-2 (PDB: 2Z7X), TLR-4 (PDB: 3FXI), and TLR-6 (PDB: 379A) was investigated using pyDockWEB (t/index) . In addiP. gingivalis is presented in The workflow to identify novel immunogenic targets against P. gingivalis consist of 1985 and 1418 proteins, respectively. Based on the KEGG mapping of core, accessory, and unique genes among different P. gingivalis strains, six different categories were introduced. The majority of core proteins were involved in metabolism, followed by genetic information processing and environmental information processing. See The core-pan plot showed that the pan-proteome and core-proteome of Surface-exposed and secreted proteins are more easily represented in the immune system and are capable of inducing a robust immune response. Thus, a total number of 39 surface-exposed proteins, including 35 Outer membrane Proteins (OMPs) and five extracellular proteins, were identified through subcellular localization analysis. Of the 39 proteins selected in the previous step, eight proteins were non-antigen. Moreover, among 31 antigenic proteins, five proteins were identified as allergens, so they were excluded from this study. Finally, 26 antigenic and non-allergenic proteins remained. PSI-BLAST analysis revealed no similarity between the human proteome and the 26 putative immunogenic proteins.The number of linear and conformational B-cell epitopes, the ratio of B-cell epitopes, and the ratio of MHC-II binding sites of the 25 proteins were determined and presented in Supplementary Data 1. The sequences of linear and conformational B cell epitopes of each protein are presented in Supplementary Data 2. The number of linear and conformational B-cell epitopes were as follows: WP_097626800.1 (4 and 6 epitopes), WP_005874477.1 (4 and 10 epitopes), WP_004585254.1 (4 and 6 epitopes), WP_004583657.1 (4 and 8 epitopes), WP_099779133.1 (4 and 8 epitopes), WP_099840460.1 (4 and 8 epitopes), WP_211599956.1 (3 and 7 epitopes), WP_012457596.1 (4 and 10 epitopes), WP_021664214.1 (4 and 8 epitopes), WP_004584259.1 (2 and 8 epitopes), WP_099780539.1 (3 and 7 epitopes), and WP_004583425.1 (1 and 9 epitopes). The surface-exposed linear B-cell epitopes of the novel immunogenic targets are shown in P. gingivalis.The linear and conformational B-cell epitopes were characterized, and the conservancy was determined and demonstrated in E. coli, in vivo). In this step, nineteen proteins were stable, while six were not. To facilitate the expression and purification of vaccine candidates in vitro, only proteins with a molecular weight < 110 kDa were selected for further study. Out of 26 antigenic and non-allergenic proteins, one protein had a high molecular weight (> 110 kDa) and was excluded from the study. Additional physicochemical properties of the proteins are listed in The selected proteins were classified into three distinct functional classes. The majority of proteins were involved in the cellular process (12/25), followed by virulence factors (10/25), and metabolism molecules (3/25). The estimated half-life of all proteins was > 10h , WP_004583657.1 (25), WP_021664214.1 (25), WP_099840460.1 (25), WP_004585254.1 (23), WP_012457596.1 (23), WP_099779133.1 (23), WP_099780539.1 (23), WP_004583425.1 (21), WP_097626800.1 (21), WP_211599956.1 (21), and WP_004584259.1 (20). See The results of CDD and EggNOG showed that the studied proteins are involved in the transport and metabolism of inorganic ions and the transport and metabolism of lipids. However, the function of two proteins (WP_004584259.1 and WP_099780539.1) was not detected. See Eight linear B-cell epitopes with conservation > 80% and antigenicity > 1 were considered to generate multi-epitope vaccines in three platforms, including FliC, LCL, and Naked chimeric protein. The Naked chimera was designed using eight selected epitopes and flexible and rigid linkers. Another multi-epitope vaccine was developed using the same epitopes on the FliC platform. Four epitopes with the greatest conservation and antigenicity were selected to generate an LCL-based chimeric protein. Epitope shuffling was performed, and finally, 24 different arrangements were developed. The antigenicity of these 24 models was evaluated, and the most antigenic arrangement was selected. Finally, three multi-epitope vaccines were achieved. The tertiary structure was validated and shown in The molecular docking results showed that all multi-epitope-based vaccines had a reasonable binding affinity to the TLR-1, 2, 4, and 6. However, FliC chimeric protein had the strongest affinity to the human TLRs. See Chronic periodontitis, a multifactorial chronic inflammatory disease due to dysbacteriosis, is characterized by the destruction of connective tissue and alveolar bone and has become the leading cause of tooth loss in adults. It affects almost 50% of the population worldwide and is one of the most common inflammatory diseases in humans .P. gingivalis is one of the bacteria involved in bacterial plaque biofilm formation and plays a vital role in the progression of chronic periodontitis. In a systematic review, the authors reviewed the literature on P. gingivalis and all demonstrated systemic implications. From their results, it appears that P. gingivalis also plays a role in the development of several systemic diseases, including rheumatoid arthritis, cardiovascular disease, and neurodegenerative diseases [P. gingivalis contributes to the pathogenesis of Alzheimer’s disease by secreting gingipains to promote neuronal damage [P. gingivalis proteins could prevent atherosclerosis [P. gingivalis minor fimbriae proteins (Mfa1), RgpA gingipain hemagglutinin domain 1 (HA1), and RgpA gingipain hemagglutinin domain 2 (HA2) were elicited protein-specific IgG [diseases . This bal damage . Therefoclerosis . In addiific IgG .in silico studies have been performed to find effective vaccines against this bacterium. For example, Khan et al. considered three antigenic and essential proteins including histidine kinase, Fe (2+) transporter, and capsular polysaccharide transport protein for vaccine design [et al. identified epitopes from Lys-gingipain (Kgp) and neuraminidase virulence factors of the P. gingivalis ATCC 33277 strain as candidate epitopes [On the other hand, several e design . FindingP. gingivalis strains and used a core proteome approach to develop a suitable vaccine candidate. The core proteome presents the most similar and common proteins between different bacterial strains. In addition, we used the quartile method of scoring in this study to find suitable targets. It should be mentioned that quartile analysis is a valuable approach in bioinformatics studies. This is because different criteria such as antigenicity, allergenicity, adhesion probability, etc. can be considered simultaneously in selecting the best vaccine candidates.With the advent of genome sequencing technology, the need to culture bacteria has diminished and been replaced by reverse vaccinology techniques. There are several predictive and analytical software programs such as Vaxign and VaxiJen that use the reverse vaccinology approach. These software programs were developed to identify potential vaccine candidates . This prP. gingivalis, only 39 proteins are exposed at the surface. Therefore, we analyzed them from different aspects to select the best putative immunogenic targets. For example, only proteins with a molecular weight of < 110 kDa were selected because proteins with such molecular weight are desirable for protein purification and in vitro evaluations [This comprehensive study has shown that of the 1418 proteins of luations . Moreoveluations . In thisluations .P. gingivalis that are non-homologous to human proteins. The results of the protein domain search showed that most of the selected proteins were involved in the transport and metabolism of inorganic ions and the transport and metabolism of lipids; the function of two proteins (WP_004584259.1 and WP_099780539.1) was not detected. However, the STRING database showed that the protein with accession number WP_004584259.1 has interactions with TonB-dependent receptors and lipoproteins. Unfortunately, no interaction was detected for WP_099780539.1, and the role of this protein remained unknown.This study presented 12 immunogenic targets and three multiple epitope-based vaccines against in vivo, and essential role in pathogenicity, make them excellent candidates for vaccine development [It was approved that several scarce nutrients, such as iron and nickel, are essential for bacterial growth. Gram-negative bacteria secrete chelators to competitively bind these nutrients from the environment. Transport of the resulting complexes into bacterial cells is mediated by TonB-dependent transporters (TBDTs), which are located on the outer membrane in Gram-negative bacteria. The properties of TBDTs, such as surface exposure, protective immunogenicity, wide distribution, inducible expression elopment , 39. Howelopment .To solve this problem, the development of epitope-based chimeric/subunit vaccines may be helpful. Epitope-based chimeric/subunit vaccines have many advantages over vaccines produced by conventional vaccinology. For example, they are inexpensive to develop, do not require microbial culturing, and can outperform many wet-lab experiments with saving time. They are a safer option because they do not contain the entire pathogen and are highly specific and stable . Of the et al. presented three vaccine candidates, including histidine kinase, Fe transporter, and capsular polysaccharide transport protein. Fe transporter was the common protein identified in the present study and its investigation. However, we did not identify histidine kinase and capsular polysaccharide transport protein. The use of different bioinformatics approaches and pipelines may justify this difference [The subtractive proteomics and reverse vaccinology approaches performed by Khan fference .P. gingivalis using reverse vaccinology, immunoinformatic analyses, and computer-aided approaches. Twelve novel vaccine candidates are proposed. Moreover, three multi-epitope vaccines were generated using Naked, LCL, and FliC platforms. Among three multi-epitope vaccines, FliC chimeric protein had the strongest affinity to the human TLRs while the LCL platform induced the highest level of immunoglobulins, cytokines, and Th1 response. The results of this study could help find an effective vaccine against this pathogen. This study might establish the fundamental of vaccine development against this pathogen to prevent periodontitis. However, experimental validation through in vitro and in vivo assays are necessary to confirm the safety and immunization of proposed vaccine candidates and multi-epitope vaccines.This study investigated the novel immunogenic targets against S1 Table(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.


Escherichia coli were also analyzed. Around 81% of patients were adults (≥18y). Of 3,200 (71%) urine samples with bacterial growth, 920 (29%) had a bacterial count of ≥1.0x105 CFU/ml indicating UTI. Women were more likely to have UTI compared to males . E. coli (51.6%) was the predominant causative pathogen followed by Streptococcus spp. (15.7%), Klebsiella spp. (12.1%), Enterococcus spp. (6.4%), Pseudomonas spp. (4.4%), coagulase-negative Staphylococcus spp. (2.0%), and other pathogens (7.8%). Both E. coli and Klebsiella spp. were predominantly resistant to penicillin  followed by macrolide , third-generation cephalosporins , fluoroquinolones  and carbapenem . Around 65% of patients tested positive for multi-drug resistant (MDR) uropathogens. A higher number of male patients tested positive for MDR pathogens compared to the female patients (p = 0.015). Overall, 71% of Gram-negative and 46% of Gram-positive bacteria were MDR. The burden of community-acquired UTI caused by MDR organisms was high among the study population. The findings of the study will guide clinicians to be more selective about their antibiotic choice for empirical treatment of UTI and alleviate misuse/overuse of antibiotics in the community.Urinary tract infection (UTI) accounts for a significant morbidity and mortality across the world and is a leading cause for antibiotic prescriptions in the community especially in developing countries. Empirical choice of antibiotics for treatment of UTI is often discordant with the drug susceptibility of the etiologic agent. This study aimed to estimate the prevalence of community-acquired UTI caused by antibiotic resistant organisms. This was a cross-sectional study where urine samples were prospectively collected from 4,500 patients at the icddr,b diagnostic clinic in Dhaka, Bangladesh during 2016–2018. Urine samples were analyzed by standard culture method and the isolated bacteria were tested for antibiotic susceptibility by using disc diffusion method and VITEK-2. Descriptive statistics were used to estimate the prevalence of community acquired UTI (CA-UTI) by different age groups, sex, and etiology of infection. Relationship between the etiology of CA-UTI and age and sex of patients was analyzed using binary logistic regression analysis. Seasonal trends in the prevalence of CA-UTI, multi-drug resistant (MDR) pathogens and MDR  Urinary tract infections (UTIs) in both community and hospital settings are estimated to affect around 405 million people globally and nearly 0.23 million people died of UTIs, contributing to 5.2 million disability-adjusted life years (DALYs) in 2019  isolates were obtained from 920 patients of which 989 were bacterial isolates and 2 were identified as 7%, 10%) . Of 71 pE. coli and Klebsiella spp. were resistant to different classes of antibiotics with the highest rate of resistance to penicillin  followed by macrolide , third-generation cephalosporins , fluoroquinolone  and sulfonamide . The difference in the prevalence of resistance in E. coli and Klebsiella spp. was significant for several antibiotics including third-generation cephalosporins, fluoroquinolones, nitrofurantoin, and sulphonamide (E. coli and Klebsiella spp. no significant variation in resistance frequency was observed between the two years (E. coli (5%) and Klebsiella spp. (9%)  were third-generation cephalosporins resistant (3GCr) and 233 (67%) of 3GCr E. coli were positive for ESBL.Among Gram-negative bacteria, honamide . For botwo years . Carbapepp. (9%) . OverallStreptococcus spp. and Enterococcus spp. were predominantly resistant to macrolide . Only a small proportion (9%) of Streptococcus spp. was resistant to third-generation cephalosporin  isolates. Among the prevalent Gram-positive organisms, both losporin . Overallp<001). Male to female ratio among laboratory-confirmed CA-UTI patients were 1:3 . E. coli was the predominant uropathogen among adult female patients followed by Streptococcus spp., Klebsiella spp., and Enterococcus spp. We observed similar trends among adult male patients except that coagulase negative Staphylococcus spp. was more prevalent than Enterococcus spp.  had an infection with both Gram-negative and Gram-positive bacteria (cus spp. .p = 0.015). The difference was prominent among child patients. Male children had 72% (CI: 22%-90%) higher odds of being infected with MDR organisms compared to their female counterparts  tested positive for MDR organisms compared to their female counterparts (63%) .We analyzed the distribution of antibiotic resistance in 991 isolates over two years to find the trends across years. There were no significant fluctuations in the prevalence of resistance to any particular antibiotic over the years among major uropathogens such as cus spp. . Overall5 CFU/ml from urine culture) was 20% with women bearing the greater burden (78%), similar to most studies of CA-UTI [Increased resistance to multiple antibiotics among pathogens causing CA-UTI leads to frequent treatment failure and complications. Drug-resistant CA-UTI has become a major global health problem, especially in developing countries like Bangladesh. However, longitudinal surveillance of CA-UTI estimating the burden of infection, etiology and antibiotic susceptibility patterns of uropathogens in Bangladesh is largely missing except for a few small observational studies among specific population groups such as pregnant women, children, and hospitalized patients , 24, 25.f CA-UTI .E. coli coupled with lactose non-fermenting E. coli or Klebsiella spp. In general, polymicrobial infections are less common in young, healthy, sexually active females [Although the majority of UTI cases were caused by a single pathogen, we found a small proportion of patients had polymicrobial growth in their urine culture mostly  females . In our  females . It is m females .Streptococcus spp. was the most prevalent cause of CA-UTI after E. coli, which is in agreement with a previous study among adult women in Dhaka slums [Staphylococcus saprophyticus was the second leading cause of UTI after E. coli [Klebsiella pneumoniae as the second leading cause [E. coli appears to be the most common cause of UTI, and thus combating E. coli infections would largely reduce the burden of CA-UTI in the community. Among all Streptococcus spp. cases S. agalactiae or Group B Streptococcus (GBS) accounted for 52% (n = 80) and around 9% of the total number of patients with UTI; 92% (n = 74) of women were infected with GBS. In a previous study among pregnant women in rural Bangladesh, only a small proportion of (5.3%) patients were infected with GBS [Among monomicrobial cases, we found that ka slums . However E. coli , 18 whilng cause . There mwith GBS .E. coli or ESBL-producing E. coli. Among neighboring countries, a study in Kathmandu, Nepal reported that 41% of bacterial pathogens from UTI cases were MDR [E. coli as 43% and 59%, respectively [E. coli isolates were MDR and 69% of isolates were resistant to third-generation cephalosporins, which is higher than the prevalence of MDR E. coli (58%) reported earlier from Bangladesh [E. coli, a significant proportion (70%) was sensitive to aminoglycosides, nitrofurantoin and carbapenem, which corroborates with data from other countries [Around 65% of the patients in our study tested positive for an MDR pathogen comprising both Gram-positive and Gram-negative organisms, which is higher than the report from a neighboring country Nepal , 32. Thewere MDR . Studiesectively , 34, 35.ngladesh . Overallountries –40.We found that male patients were more likely to be infected with MDR organisms than female patients. Similarly, prevalence of 3GCr and carbapenem-resistant organisms was higher among adult male compared to adult females. This finding correlates with the results of a large study conducted in Portugal where the prevalence of MDR uropathogens was higher in men than in women (35% vs. 22%) . HoweverThere were some limitations in our study. We carried out the surveillance among patients who came to icddr,b diagnostic facility to test their urine samples for culture and sensitivity analysis. A large proportion of patients receiving diagnostic services from icddr,b are the residents of Dhaka city and therefore the data may not be representative of the whole country. icddr,b diagnostic facility only provides clinical diagnostic services and does not offer any outpatient services and therefore, we could not estimate the proportion of patients with and without symptomatic infection. Nevertheless, our study provides a recent insight into the etiology of CA-UTI along with trends in antibiotic resistance among uropathogens over 2 years in a large sample size for the first time in Bangladesh.The burden of CA-UTI caused by MDR pathogens is high among the study participants and there was no significant difference in prevalence and etiology of infection over two years. The findings of the study can serve as an evidence base for updating guidelines for improved management of CA-UTI in Bangladesh and other developing countries with similar settings. It also provides a basis to conduct epidemiologic studies to investigate risk factors for the acquisition of antibiotic-resistant CA-UTI.S1 Data(XLSX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.