Tomato peel/pulp nsLTP, Sola l 3, was degraded after digestion,although the immunoglobulin E (IgE) recognition of intact proteinand a 10 kDa band were still observed after 30 min of duodenal digestionin the presence of phosphatidylcholine. The tomato seed nsLTP, Solal 7, showed a higher stability than the other seed allergen, Solal 6, during digestion. Sola l 7 showed an IgE immunoreactive 6.5 kDaband in immunoblotting analysis, retaining up to 7% of its IgE-bindingcapacity in inhibition ELISA test after 60 min of duodenal digestionand keeping intact its ability to activate basophils after digestion.These results suggest that the tomato seed allergen Sola l 7 mightbe considered as an important allergen in the induction of allergicresponses to tomato due to its high stability against gastrointestinaldigestion.Theinfluence of gastrointestinal digestion on the immunologicalproperties of three different nonspecific lipid-transfer proteins(nsLTPs) described in tomato fruit has been assessed using an  In thisregard, the tomato (Solanum lycopersicum), belonging to the Solanaceae family,has been described as one of the most prevalent plant-derived foodsensitizers.8 In the allergogram of tomato fruit, five allergensfrom the peel and pulp, Sola l 1–Sola l 5, have been identifiedand characterized to date,11 and other proteins such as Solal Glucanase and Sola l Peroxidase have also been detected, althoughthey need deeper characterization.Pru p 3, thensLTP from the peach peel, is considered the mainsensitizer in nsLTP allergy among the 12 In this context, we have previously shown thattomato seed extract recruited a significant IgE reactivity, and weidentified two new tomato seed allergens, Sola l 6 and Sola l 7, responsiblefor severe symptoms (anaphylaxis and angioedema) in the studied population.13 Both proteins differ structurally and immunologicallyto the already described tomato peel/pulp nsLTP, Sola l 3.13 Sola l 3 and Sola l 7 belong to class 1 nsLTPs,to which Pru p 3 also belongs, being identified in a multitude ofplant sources, trees, weed pollens, and foods and characterized bya long tunnel-like cavity. In contrast, Sola l 6 belong to class 2nsLTPs, which are proteins with two adjacent hydrophobic cavities,are scarcely studied, and are neither very abundant nor have a highallergenic potency in the few sources described.14Recently, numerous studieshave focused on the role of seed allergensin triggering severe and unexpected allergic reactions.17 Moreover, nsLTPs can bind lipids through their hydrophobiccavity, which is a key point in the maintenance of allergenicity duringthe digestion of certain proteins. The interaction of lipids withproteins in solution has been demonstrated to be important in theaffecting rates of proteolysis and in particular has provided a protectiveeffect for some milk proteins.19Food allergens are usually resistant to the harshacidic environmentof the stomach and resist digestion by gastrointestinal enzymes. Therefore,they can reach the gut-associated lymphoid tissues with still a highimmunologic potency.21 Because of the difficulties involvedin conducting in vivo experiments to evaluate thegastrointestinal stability of the different food allergens, in vitro digestion models provide an alternative tool.23On the contrary, allergens can be digested into molecularmasspeptide fragments big enough to retain the IgE-binding and T-cellstimulating capacity.in vitro simulated gastrointestinal digestionwas performed using a two step system, which mimics the successivepassage through the stomach and duodenum and includes phosphatidylcholine(PC), a physiological surfactant secreted by the stomach mucosa.The aim of this work was to provide an immunological characterizationof the digestion products of three tomato nsLTPs from the peel andseeds by immunoassays and basophil activation test (BAT). For thispurpose, an 13 Natural tomato allergens were used for skin prick test.Natural Solal 3, Sola l 6, and Sola l 7 were purified from Applause tomatoes andcharacterized as described previously.E. coli cells). Subsequently,sequences were cloned into the pPICZαA vector and electroporatedinto Pichia pastoris KM71H electrocompetentcells. After 72 h of methanol induction, recombinant tomato proteinswere isolated from extracellular culture and purified throughout aDEAE anion exchanged chromatography and a C-18 reverse phase columnanchored to a reverse phase high-performance liquid chromatograph(RP-HPLC), with the same conditions as those described for the naturalones.13 Protein concentration was determinedby a bicinchoninic acid protein assay kit  and spectroscopic analysis. The maintenance of the secondary andtertiary structures of recombinant allergens compared with those ofthe native counterparts and their thermal stability was verified bymeans circular dichroism  andnuclear magnetic resonance experiments (data not shown).Inorder to produce the recombinant form of tomato allergenic nsLTPs,we isolated total RNA and with specific primers designed from thefingerprint analysis; we cloned the specific fragment into the PCR2.1 vector  to tomato together with evidence of specific IgE antibodies(sIgE). The SPT with nsLTP  was also performed to assess the LTP sensitization. The patientswere classified according to their clinical symptoms after tomatointake: mild reactions, including oral allergic syndrome, moderatereactions, such as urticaria, and severe reactions, including anaphylaxis1.24 Moreover, homemade pulp andseed tomato extracts were also used at 5 and 50 μg/mL for totalprotein concentration. Histamine dihydrochloride (10 mg/mL) and physiologicsaline solution were used as positive and negative controls, respectively.SPT responses were read after 15 min, and a wheal size of 3 mm2 greater than the negative control was considered positive.SPT with tomato extract and purifiedtomato seed nsLTPs described in this study  at 5 and 50 μg/mL were conducted according to standardprocedures.The total tomato sIgE levels in serum samples were determined byImmunoCAP-FEIA according to manufacturer’s instructions  1.A group of age- and sex-matchedindividuals with a tolerance totomato and without food allergic symptoms was included as a negativecontrol.All human samples were obtained according to the principlesofthe Declaration of Helsinki and approved by the Ethics Committee ofboth hospitals. All patients signed the corresponding informed consents.The Ethical Committee of the Complutense University and the Ethical and Research Committee of Hospital Regional Universitarioof Malaga (#02/2012) approved the protocols used for this experimentalwork and all the methodology related to the use of human sera in thisstudy.In vitro gastroduodenal digestions of recombinantSola l 3, Sola l 6, and Sola l 7, using simulated fluids, were performedas described previously.25 Briefly, gastricdigestion (GD) was conducted in simulated gastric fluid  in the absence or presence of phosphatidylcholine (PC) vesiclesat 37 °C, with the addition of porcine pepsin (182 U/mg of protein).Phospholipid vesicles were prepared by dissolving egg l-α-phosphatidylcholinefrom Larodan  in 35 mM NaCl pH 2.0 at a concentrationof 9.58 mg/mL. Then, the solution was sonicated in ice ,not exceeding a sample temperature of 40 °C. Phospholipid vesicleswere filtered through Filtropur 0.45 μm of poly(ether sulfone)from Sarstedt  to remove any possible titaniumparticles. During gastric digestion, aliquots were withdrawn at 0,15, 30, and 60 min. Duodenal digestion (DD) was performed on the 60min gastric digests readjusted to pH 6.5, with the addition of 0.25M Bis-Tris, pH 6.5, 1 M CaCl2, and a 0.25 M bile saltsmixture, containing equimolar quantities of sodium glycodeoxycholateand sodium taurocholate. After preheating at 37 °C for 15 min,pancreatic porcine lipase (24.7 U/mg protein) and colipase , pancreatic bovine trypsin (34.5 U/mg protein), and α-chymotrypsin(34.5 U/mg protein) were added. The reactions were stopped after 30and 60 min at 37 °C by adding a solution of Bowman–Birkinhibitor. All enzymes and reagents were purchase from Sigma-Aldrich.Native tomato nsLTPs and their gastroduodenal digestswere analyzedin a RP-HPLC SHIMADZU (LC 20AB) system using an Ultrasphere reverse-phaseC-18 column. Operating conditions were as follows: solvent A, 1 mL/LTFA in Milli-Q water; solvent B, 1 mL/L TFA in HPLC grade acetonitrile;flow rate, 1.5 mL/min; injection volume, 200 μL (1 mg/mL). Alinear gradient of solvent B in A, from 0% to 30% in 60 min, followedby 50% B for 20 min was used.26 using pooledsera from five , with5% (v/v) of β-mercaptoethanol (β-ME) and were heated at95 °C for 5 min. Samples were loaded and analyzed on PrecastCriterion XT 16.5% Tris-Tricine gels (Bio-Rad). Separations were carriedout at 100 V during 1 h and 45 min, using a Tris-Tricine running buffer(Bio-rad) in the Criterion cell. Finally, gels were stained with Bio-SafeCoomassie G-250 (Bio-Rad).12 Briefly, the binding of human IgE was detected by usingthe mouse anti-human IgE antibodies (diluted 1:5000) kindly donatedby Alk-Abelló, followed by horseradish peroxidase (HRP)-labeledrabbit anti-mouse IgG (diluted 1:5000) .The signal was developed with a chemiluminescent ECL-Western blottingreagent .After SDS-PAGEseparation, gelswere soaked in transfer buffer  for 20 min and subjected to semidry transfer in a Trans-BlotSD (Bio-Rad) for 30 min at 18 V. The nitrocellulose membranes wereblocked with 3% skim milk powder in phosphate-buffered saline, pH7.4 containing 0.05% Tween 20 PBS-T. The IgE immune detection of theallergenic fragments after gastrointestinal digestions of Sola l 3and Sola l 7 were carried out with pools of five (numbers 1–5from 2 (w/v), 0.033%MgCl2 (w/v), 0.1% HSA (w/v), 10 μL/mL of IL-3 (1mg/mL), and 1 μL of monoclonal antibody CCR3-APC (1 mg/mL)  for 10 min at 37 °C. Then, 100 μL ofeach purified Sola l 7, Sola l 6, and their digests was added at differentconcentrations following a 10-fold dilution pattern (0.1–0.00001μg/mL) and incubated for 30 min at 37 °C. Anti-human IgE(0.5 mg/mL)  or PBS were usedas a positive and negative controls, respectively. After 5 min onice to stop the degranulation process, samples were incubated with1 mg/mL of monoclonal antibodies anti-CD 203c-PE and CD63 FITC (BioLegend)for 15–20 min at 4 °C. Finally, lysis solution (BD Biosciences)was used for red cells disruption. Cells were analyzed using a FACSCaliburflow cytometer (BD Biosciences), acquiring at least 500 basophilsper sample. Results are presented as the percentage of activated basophils(CD63+CD203c+CCR3+). Due to the lowavailability of patients, only the sera of one tomato allergic patientsensitized to Sola l 6 and Sola l 7 and displaying anaphylaxis  was used for this experiment.The basophil activation testwas performed using 100 mL of heparinized whole blood, incubated with20 μL of stimulation buffer (1 M HEPES buffer containing 0.78%NaCl (w/v), 0.037% KCl (w/v);, 0.078% CaClFigure S1). The three proteins presented thetypical characteristics of α-helix-enriched proteins, as nsLTPsare, with a secondary structure showing two clearly marked minimaat 208 and 220 nm.Inorder to demonstratethe similarity of the recombinant proteins with their natural analogues,the content in the secondary structure of purified recombinant proteinswas spectroscopically determined by circular dichroism (CD) . Interestingly, the recombinant formwas produced in the same trimeric form by the Pichia yeast, showingthe same degree of recognition by a pool of sera from allergic patients.Sola l 7 is produced inthe seeds as a trimeric form, showing thesame IgE recognition by sensitized patients as the monomeric one whenwas treated with a reducing agent, such as β-ME (in vitro gastric (GD) and duodenal (DD) digestions are shownin in vitro GD was performed in the absence and presence ofPC, a physiological lipid component secreted by the gastric mucosaand also present in the bile during digestion. On the basis of proteomicresults, tomato peel nsLTP, Sola l 3, did not show significant changescompared to undigested protein during GD  conformation of class 1nsLTPs,and the presence of an extensive disulfide bond core, makes theseallergens exceptionally resistant to thermal or enzymatic degradation,and it is thought to be one of the most important factors to maintainthe 3D structure of a protein contributing to the severe systemicreactions often observed in allergic patients.Inorder to compare the IgE reactivity of digestionproducts, samples were evaluated by direct immunoblotting using apool of sera from tomato allergic patients. Due to the limited amountof serum available for the other tomato nsLTPs, we selected digestedsamples from Sola l 7 among seed nsLTPs based on its higher resistantto digestion. The immunoblotting of gastric and duodenal digests inthe absence and presence of PC is shown in The IgE binding of Sola l 3 felldrastically at the duodenal phase in the absence of PC. However, after30 min of DD in the presence of PC, the IgE recognition of intactprotein is still observed as well as a 10 kDa band 2A. Intac37 and it has been described that the partial unfolding under the acidicgastric conditions, could enhance an effective allergic response,likely because it unmasks B-cell epitopes.38 In agreement with immunoblotting data, IgE reactivity fell drasticallyat the end of DD until values close to zero when the digestion wasperformed in the absence of PC. When PC was included in the simulateddigestion, the IgE reactivity remained close to 13%, compared to theundigested protein, showing the protective effect of PC on the enzymaticdegradation of Sola l 3.We further checked the samplesby inhibition ELISA with the samepool of sera as in the immunoblotting. The immunoreactivity of Solal 3 increased at the end of the GD, showing values of 185% of IgEbinding in the absence of PC and even higher in the presence of PC,reaching values of 241% compared to the undigested protein and indicatingthat the unfolding of the protein during the gastric phase would exposeIgE epitopes 2C. It is39 Examples are the easily digestible pollen-related food allergens,such as Mal d 1 (apple), Api f 1 (celery), and Cor a 1 (hazelnut)belonging to Bet v 1-like family, which completely lose their abilityto bind IgE in the first minutes of GD/DD. As it has been shown withSola l 3, the presence of PC during digestion exerted a protectiveeffect against the degradation of Sola l 7, preserving immunoreactivityvalues against IgE at the end of DD of up to 37% compared to thatof undigested protein.In contrast, Sola l 7 retained 7% ofIgE-binding capacity aftergastroduodenal digestion in the absence of PC 2D despit40 preserving almost the whole core of the moleculeas an immunogenic fragment, as observed at the end of the DD of Solal 7 .The structural compactness of nsLTPsis attributed to the presenceof a conserved skeleton of cysteine residues that forms multiple disulfidebonds. However, nsLTPs allergens also present type 1 (linear) epitopes,according to ample literature consensus. This composition gives theseallergens excellent properties to resist digestion and heat treatment,ex vivo by performing a basophil activation test (in vitro digestibilityand allergenicity of certain allergens has been reported,41 maybe because the in vitro digestibilityof a protein is influenced by the hydrolysis conditions and enzymequantities, not considering the protein interactions with other digestiveor food matrix components. In addition, even though certain proteinsare consistently degraded in the in vitro assays,it cannot be discarded that, in an in vivo situation,minimal amounts of intact material in an immunologically active formbypass the digestion.42 In our case, thediscrepancies found between the in vitro studiesperformed on Sola l 7, with a maximum of 7% IgE binding, and in ex vivo experiments may be due to the fact that the secondtechnique is more sensitive and the degradation products are in liquidphase, being exposed all the IgE epitopes of the protein, either conformationalor linear ones. For this reason, a complete study at different levelsis essential for this type of approach.To study,in-depth, the repercussion of the digestion on the allergenicity oftomato seed allergens, Sola l 6 and Sola l 7, and in order to corroboratethe data obtained from the immunoblotting and the ELISA for Sola l7 digests, the allergenic capacity of the intact proteins and theirdigests obtained in the presence and absence of PC was evaluated ion test 3. The abin vitro gastrointestinal digestionof three nsLTPs described in tomato fruitwith a high allergenic potential. Tomato peel nsLTP, Sola l 3, showedpartial resistance to gastrointestinal digestion (In summary, we provided for thefirst time the igestion 1A,B, whi13 The in-depth characterization carried out inthis work will allow for advancement in the knowledge of tomato seedallergens, of which their use should be considered in clinical diagnosisand to establish consumption patterns for tomato allergic patients.In this manuscript,we suggest that the tomato seed allergen Solal 7 might be an important allergen to be considered during allergicsensitization and/or allergic responses to tomato due to its highimmunogenicity and stability against gastrointestinal digestion. Todate, only allergens from the tomato fruit have been reported. Theidentification and characterization of two new seed allergens couldfacilitate the tomato allergy diagnosis. Usually, only the pulp isapplied to carry out the skin prick test used as a diagnostic test,and therefore, some patients reactive to the seed proteins could beconsidered as false negatives. The inclusion of Sola l 7 in diagnostictests could improve the diagnosis of tomato allergy since seeds areincluded in many processed foods, and tomato seed allergens, as Solal 7, are described as responsible for anaphylactic shock during ingestion.


We combined experimentalassays with molecular simulations to investigate this IRI agent, drawingcomparison to β-alanine, an isomer of l-α-alaninewhich displays no IRI activity. We found that the difference in theIRI activity of these molecules does not originate from their icebinding affinity, but from their capacity to (not) become overgrown,dictated by the degree of structural (in)compatibility within thegrowing ice lattice. These findings shed new light on the microscopicmechanisms of small molecule cryoprotectants, particularly in termsof their molecular structure and overgrowth by ice.Extremophiles producemacromolecules which inhibit ice recrystallization,but there is increasing interest in discovering and developing smallmolecules that can modulate ice growth. Realizing their potentialrequires an understanding of how these molecules function at the atomisticlevel. Here, we report the discovery that the amino acid  A major contributorto post-thaw damage is ice recrystallization (IR), an Ostwald ripeningprocess7 whereby large ice crystals growin favor of smaller ones, exerting mechanical and osmotic stress onthe sample which often leads to post-thaw cell death. Ice recrystallizationinhibition (IRI) represents an appealing strategy to improve cryopreservationoutcomes, and ice recrystallization inhibitors (IRIs) have consequentlyreceived significant interest in recent years. However, the mechanism(s)by which these molecules inhibit IR and the underlying molecular determinantsare poorly understood. In turn, this hampers the discovery of newmaterials that possess a sought-after balance of potent inhibition(at low concentrations), biocompatibility, and amenability to lowcost, large scale production.The cryopreservation of biologicalmaterials is a key factor in regenerative medicine and cell-basedtherapies,8 to the well-known ice-bindingproteins,9 to small molecules.10 These materials can all give rise to the samemacroscopic effect defined as IRI, but multiple molecular-level mechanismsappear to underpin the observable phenomenon. For certain materials,including many of the antifreeze proteins (AFPs) and glycoproteins(AFGPs), IRI activity is linked to a molecule’s ability tobind to specific faces of an ice crystal, causing a local positivecurvature of the ice front which inhibits further ice growth throughthe Gibbs–Thomson (Kelvin) effect.12 By arresting ice growth, surface adsorption also results in a non-colligativedepression of the freezing point relative to the melting point, knownas thermal hysteresis (TH). TH is often accompanied by dynamic iceshaping (DIS), whereby ice crystals display distinct morphologiesreflecting the specific lattice face(s) onto which the AF(G)Ps adsorbed.13 The question remains: how do these materialsfirst recognize and bind ice in a vast excess of liquid water? Theanswer is still unclear. A diversity of chemical motifs appear capableof binding to ice, but the relative contributions of hydrogen bonding,hydrophobic interactions,15 and ordered clathratewaters16 are still under debate.The IRI-active materials discoveredto date are diverse, rangingfrom polymers,18 Meanwhile, for other IRI-active materials such as the small moleculecarbohydrates reported by Ben and co-workers,20 there is no evidence of ice binding at all .These findings both point to an alternate mode of inhibition thatis independent of ice binding and likely the primary mechanism ofaction for small molecules. One possible mechanism, proposed by Benand colleagues,19 suggests that small moleculesinhibit IR by disrupting the order of water in the interfacial regionbetween bulk water and the preordered water layer surrounding an icecrystal. However, this hypothesis lacks extensive experimental supportand is challenging to study computationally. Given that ice bindingcan often shape bound crystals into needlelike spicules that are injuriousto biological materials, understanding this mode of inhibition representsa crucial step toward the identification of novel IR inhibitors whichhave clinical application. In this context, small molecules are alsoattractive targets because they can be efficiently manufactured, andobtaining structure–property relationships is simpler thanfor, e.g., polymers, which have intrinsic heterogeneity .Equally unresolved are the mechanistic details of IRI. Recent studieshave confirmed that, although IRI and TH activities appear connected,the two properties are not directly correlated in AFPs.l-α-alanine exhibits IRI activity at millimolarconcentrations. We also show that its structural isomer, β-alanine,is IRI-inactive at equivalent concentrations, despite the structuralsimilarity of these two forms. To unravel the origin of α-alanine’sIRI activity we used atomistic molecular dynamics (MD) simulations.These simulations allow for ice growth kinetics to be studied in thepresence of (α/β-)alanine, shedding new light on the mechanismsof small molecule IRIs and the molecular determinants of IRI activity.Here, we report that the simple amino acid 22 When α-alanine was tested in phosphatebuffered saline (PBS), which is typically used for this assay, noIRI activity was observed .However, in a series of controls using α-alanine and betaine, we found that including 10 mM NaCl led to significantIRI with α-alanine (and not betaine) and is a sufficient quantityof saline to avoid false positive hits .23 We also found that α-alanine remains active in 10 mM phosphatebuffer but not under higher concentrations of saline (100 mM NaCl); hence, we suggest that theIRI activity of this system is sensitive to high concentrations ofsalt rather than specific components of the PBS solution. We alsonote that, while this assay is usually performed in PBS, the use ofsaline solution for IRI measurements is not uncommon in this field.25We began by assessing the ability of α- and β-alanineto inhibit IR using the “splat” cooling assay. Thisassay involves rapidly cooling a droplet of solution to form a polycrystallineice monolayer 1c, which26 but on the same magnitude as other small moleculestested under similar conditions.24 Forexample, PVA20 (where 20 is the degree of polymerization) achievessimilar levels of inhibition to 100 mM α-alanine (∼10mg/mL) at ∼1 mg/mL.27 However, theIRI activity of α-alanine must be considered in the contextof the molecule’s size. Given that its structure comprisesmerely 13 atoms (corresponding to a molecular mass below 90 atomicmass units), to our knowledge α-alanine represents the smallestice recrystallization inhibitor discovered to date; it is significantlysmaller than other materials  reportedelsewhere.28Using 10 mM NaCl, we found that α-alanine is able to suppressice growth almost entirely at millimolar concentrations, producingcrystals which are dramatically smaller than in the controls 1a. This 29 M9,30 TB,31 and MK32 media),and such materials require effective cryopreservation for both industrialand research applications.In the knowledge that α-alanine is inactive at highsaltconcentrations (100 mM and above), we note that the cryoprotectiveapplications of α-alanine specifically may be limited to caseswhere biological materials are stored in low-salt buffers. However,we highlight that there are a number bacterial and plant growth mediawhich satisfy these minimal salt requirements . This is reminiscent of other small molecule IRIs elsewhere discovered,19 in contrast to larger IRI-active materials .35 In the standard “sucrose-sandwich”assay, we also observed no ice growth inhibition . We hypothesize that this is due to the high fractionof liquid (sucrose solution) in this assay, which means that diffusioneffects are dominant and the effective concentration of amino acidat the ice/water interface is very low, resulting in no observableactivity.To investigate whether α-alanine hasany effect on ice crystalmorphology, and therefore binds ice, we used a modified version ofthe “sucrose-sandwich” assay. In this assay, concentratedsucrose solution (50% w/v) is used to produce segregated ice crystalswhose individual shapes can be clearly observed. Crystals grown inthe presence of α-alanine did not exhibit morphologies thatare characteristic of ice-binding  is exposed to the water in the xy-plane, seeding ice growth in the ±z-direction. We used the all-atomistic CHARMM36 force field36 along with the TIP4P/Ice model37 to simulate the amino acid and water molecules. Furtherdetails of the computational setup can be found in the Supporting Information. Using this setup, wecollected 60 statistically independent trajectories for α-alanineas well as β-alanine: 20 for each of the prismatic and the basalfaces. We also probed concentration effects by including either oneor two molecules in each of the two water slabs in every trajectory.The simulations were run for 100 ns (prismatic faces) or 120 ns , by which time only a small quantity of liquid water typicallyremains in the simulation cell. We quantified ice growth by calculatingthe number of molecules in the seeded ice cluster over time, whichwas then converted to a growth rate. We note that while these simulationsdo not fully capture the complex recrystallization process  in its entirety, the transfer of water molecules from thesupercooled liquid fraction at the grain boundaries to the surfaceof a growing crystal is a fundamental step in ice recrystallization.Our simulations capture the kinetics of this process , which is thus directly proportional to the rate of ice recrystallizationrepresented by the % MGS metric. This computational methodology hasbeen validated extensively, and results obtained via this setup haveshown excellent correlation with the experimentally observed IRI activityof both polymers38 and small peptides.39In the absence of ice shaping effects, we looked toidentify analternative mechanism to explain the IRI activities of α- andβ-alanine that does not depend on ice binding. This amountsto a challenging task, given the transient nature of the ice/waterinterface during recrystallization and the size and similarities ofthese two structures. To overcome this, we used atomistic MD simulationsto study the growth of ice in the presence of α- or β-alanine.These simulations comprise an ice/water interface featuring a centralslab of ice and two adjacent slabs of water in contact with a vacuum,as depicted in 40 To quantify these results categorically, we define a simulationas showing ice growth inhibition when the observed growth rate isbelow 0.03 m/s. This corresponds to a minimum of 50% reduction inthe growth rate compared to our control simulations which containno (α/β-)alanine molecules, although the following trendshold regardless of the chosen cutoff value within the range of 0.01and 0.06 m/s. Applying this definition, we observed 5 and 6 instancesof inhibition by α-alanine, and 1 and 2 instances by β-alanine,for the primary and secondary prismatic planes, respectively alanineis deposited under two or more layers (∼8 Å) of ice bythe end of the simulation. These outcomes, summarized in 42With our simulation and experimentaldata in agreement, we examinedthe trajectories looking for differences between α- and β-alaninethat could explain their IRI activities. Interestingly, a significantnumber of (α/β-)alanine molecules were found to becomeovergrown by the advancing ice front and then incorporated into thelattice, as depicted in Figure S5). We also suggest that the overgrowth of (α/β-)alaninemolecules has a compounding effect, as it sequesters the amino acidsin the ice fraction. Consequently, the effective concentration ofα-alanine at the grain boundaries could increase over time relativeto β-alanine, further slowing ice growth via greater surfacecoverage. It is necessary to point out that there are cases whereinindividual simulations, as with their experimental counterparts, donot reflect this overall trend. Fundamentally, IRI activity is notan “on/off” property, especially when examined at thescale of these simulations, and a large number of independent trajectorieswere therefore required to validate these findings. We also note thatin the simulations where each ice front is exposed to two (α/β-)alaninemolecules, the same trends in terms of ice growth inhibition and overgrowthare observed . In these simulations, icealso grows at a consistently slower rate compared to those containinga single amino acid molecule , reflecting the concentration effects we observe experimentallyalanine molecule is not overgrown, it is still ableto disrupt the growth of ice at or near the interface. Indeed, suchdifferences can clearly be observed by comparing the growth ratesof nonovergrown and overgrown trajectories S5. We al44 Our previous work38 also revealed that, for the flexible polymerPVA, the effective volume and contact area with the ice surface canalso determine the strength of IRI. Therefore, we first computed thevolume and solvent-accessible surface area (SASA) occupied by thesetwo molecules throughout the course of the simulations. However, wefound that these properties of α-alanine and β-alanineare almost indistinguishable, with average volumes and surface areasdiffering by approximately 1 Å3 and 3 Å2 in volume and SASA, respectively, corresponding to a relative differenceof around 1% for each quantity . Moreover, while α-alanine typically occupies a larger volumethan β-alanine, this trend is reversed with respect to the surfaceareas. Hence, molecular volumes or surface areas do not appear tobe correlated with IRI activity for these small molecules. We alsoinvestigated the (binding) orientation of the molecule with respectto the ice front but found no correlation between this property andIRI activity (data not shown).To understand why β-alanineis more likely to become overgrowncompared to α-alanine, we sought to identify relevant characteristicsthat differ between these two molecules. For AFPs, the size of themolecule or area of its ice binding site is known to correlate stronglywith antifreeze activity.Figure S8, left panel). In fact, we observedthat α-alanine typically forms just one or two hydrogen bondsout of a possible four. The number of hydrogen bonds formed via theamine group, meanwhile, does not appear to differ between these twocompounds . Thisdifference arises from the relative positions of the carboxylate andamine groups in these two molecules. For α-alanine, these groupsare bound to the same (α-)carbon atom, whereas for β-alaninethey are separated by an additional methylene group . The highenergy barrier that exists between these conformers prohibits freerotation about the C– C bond, consistent with the narrow dihedraldistribution observed in our unbiased simulations . Hydrogen bond formation is sterically hinderedin these conformations, limiting the hydrogen bond interactions betweenα-alanine and water. In contrast, the free energy landscapeof β-alanine features conformational energy barriers on thesame order as thermal fluctuations at room temperature, allowing thisphase space to be fully explored during the unbiased run and greaterhydrogen bond formation compared to α-alanine.Next, we analyzed the hydrogenbonding interactions between thesemolecules and water/ice. We found that the hydrogen bonding capacityof α- and β-alanine differ significantly, consideringboth molecules share the same hydrogen bond donor and acceptor groups.Whilst β-alanine frequently forms three or four hydrogen bondswith water via its carboxylate group, the ability of α-alanineto form a full complement of bonds via this same group is impairedalaninethat are able to participate in hydrogen bonding (nitrogen and oxygen).We found that the nitrogen–oxygen distances provide a closematch with the lattice distances of the prismatic and basal planesfor β-alanine but not for α-alanine  was replacedby an oxygen atom (OAla) or nitrogen atom (NAla) from β-alanine . In contrast,the angles formed between the oxygen atoms of α-alanine showeda broader distribution and a greater deviation from the O···O···Oangle in pure ice. Similarly, the geometry of (α/β-)alaninemolecules that become overgrown (right panels, Further, we also observed that β-alanineoffers a more compatible“fit” within the crystal lattice than α-alaninewith respect to its tetrahedral arrangement with neighboring watermolecules. When a given water oxygen (O-alanine 4b, the c-alanine S10. In c-alanine S10. In c15 highlighting the differentstructural determinants at play in small molecule IRIs. These findingsprovide new insights and avenues for the discovery and developmentof small molecule cryoprotectants, building upon the ubiquitous aminoacid scaffold. In light of the limited IRI activity of α-alanineunder high salt concentrations, the identification of saline-stableinhibitors represents a focal point for future work.In summary, we have shown via α-alanine that very smallmolecules,with fewer than 15 atoms, can be effective IRI agents and representa scaffold to understand structure-function relationships. We havealso brought to attention amino acids as a new class of IRI-activematerials, investigating the IRI activity of α-alanine alongsideits isomer β-alanine, using quantitative experimental measurementsand atomistic molecular simulations. Surprisingly, we found that thedifference in IRI activity of these structures is dictated by theirpropensity to become engulfed and irreversibly overgrown by ice, underpinnedby their compatibility to fit within the ice lattice. We note thatthe trends observed here with respect to ice binding significantlydiffer from those reported in the literature for, e.g., antifreezeproteins,


The Cystic Fibrosis Foundation Patient Registry (CFFPR) reports a high prevalence of asthma (34.6%) in people with Cystic Fibrosis (PwCF). While our current understanding of this relationship is limited, a type 2 inflammatory (T2) phenotype has often been identified in CF patients.This study aimed to evaluate the relationship between the eosinophilic CF T2 inflammatory phenotype and CF-related pulmonary outcomes and microbiological data.We conducted a retrospective chart review of adult patients with CF  receiving their care at University of Virginia Medical Center adult program from January, 2013 through December, 2018. Data collected included demographic data, CFTR (CF transmembrane conductance regulator) mutation, CF comorbidities, medications, Absolute Eosinophil Counts (AEC) in cells/µL and Immunoglobulin E (IgE) levels in IU/mL.Pseudomonas spp., MRSA, other bacterial spp., Aspergillus spp., and other fungi (p < 0.001). Univariate analysis demonstrated that people with CF who experienced more than 2 exacerbations requiring hospitalizations and/or intravenous antibiotics a year were more likely to have high AEC (p = 0.018). Logistic regression showed that as AEC increases, the probability that the measurement was taken during a CF exacerbation increases (p = 0.0039). A linear mixed model showed that each additional annual exacerbation event increased on average the log IgE by 0.04. (p = 0.015). This finding remained stable in a multivariate model (p = 0.0145). When adjusted for atopy, log IgE increases as the number of exacerbation events increases (p = 0.022). There was no association between AEC and IgE and microbiological colonization.Of 93 patients screened for study eligibility, 74 were included in the final analysis; 19 patients were excluded due to lack of longitudinal data across the study timeline. Lung function decline correlated with increased AEC (p < 0.001) and IgE (p < 0.001) even when adjusting for covariates: age, gender, presence of This study has shown that in CF patients, T2 inflammation based on serum AEC and IgE correlated with pulmonary exacerbations requiring hospitalizations and/or intravenous antibiotics, independent of bacterial airway colonization. In addition, lung function decline correlated with increased IgE and AEC. Further studies are needed to explore these correlations and potential impact on treatment.The online version contains supplementary material available at 10.1186/s13223-023-00760-z. The Cystic Fibrosis Foundation Patient Registry (CFFPR) reports a high prevalence of asthma 34.6%) in people with Cystic Fibrosis (PwCF) . While o4.6% in pAspergillus fumigatus, with higher levels of IL-13 and IL-14 [Pseudomonas aeruginosa infection, with a T2 inflammatory phenotype being an independent risk factor for infection [This type 2 inflammation is characterized by high total IgE antibody titers, specific IgE sensitization and eosinophilia. Also central to this signature are type 2 cytokines (interleukin (IL)-4, IL-5, and IL-13) and type 2 cytokine producing cells (in airway or sputum), such as type 2 innate lymphoid cells . CF mousnd IL-14 . Moreovend IL-14 . This T2nfection .This study aimed to evaluate the relationship between the eosinophilic CF T2 inflammatory phenotype and CF-related pulmonary outcomes and microbiological data. As there are many parameters for T2 inflammation, we focused primarily on absolute eosinophil count (AEC) and IgE levels. We hypothesized that higher levels of AEC and IgE would be associated with poor lung function (FEV1), increased frequency of CF exacerbations, and higher susceptibility to microbiological infections.We conducted a retrospective chart review of adult patients with CF  receiving their care at University of Virginia Medical Center adult program from January, 2013 through December, 2018. Inclusion criteria were diagnosis of CF, and complete data in our medical system for at least 3 of the 5 years. Overall, 19 patients were excluded from the study due to incomplete data during the study period, due to having less than one annual visit at our center, undergoing lung transplantation during the study period, moving to a different center during the study period, or being new to our center in the period, due to transitioning from pediatric or moving to our center from a different center.Mycobacteria sppc, Methicillin Sensitive and Resistant Staphylococcus Aureus (MRSA), Pseudomonas Aeruginosa, Stenotrophomonas spp., Achromobacter spp., Aspergillus Fumigatus and other Aspergillus spp., Exophiala spp., Rasamsonia spp., and Scedosporium spp. Fungal and bacterial presence was defined as detection of a positive fungal or bacterial culture of patient sputum, respectively.Patient characteristics collected included demographic data , CFTR mutation, CF comorbidities , Asthma), medications , Absolute Eosinophil Counts (AEC) in cells/µL  and total Immunoglobulin E (IgE) levels in IU/mL  and specific respiratory or fungal Immunoglobulin E panels. Asthma diagnosis was collected in the study data if it was documented by the clinical team in the electronical medical record at any time during the study period. PpFEV1 (percent predicted Forced Expiratory Volume in 1 s) and rate of pulmonary exacerbations  were retrospectively collected. The severity of CF disease based on mean ppFEV1 was determined at the end of the study period, in 2018. The CF severity was defined as normal if ppFEV1 was 90% or higher. Mild CF disease was defined as ppFEV1 between 70 and 90%, moderate as ppFEV1 of 40–90% and severe disease if ppFEV1 was below 40% . Mean luExophiala spp., Rasamsonia spp., and Scedosporium spp. as (E/R/S) due to their small sample size as we conjectured that it would be important to include them in our analyses since those fungal groups are known to cause disease in CF patients. Similarly, Achromobacter spp. and Stenotrophomonas spp. were grouped together.AEC and IgE were considered high if they were above 300 cells/µL and 180 IU/mL respectively. We also grouped together This study was approved by our institution’s Institutional Review Board Human Subjects Research (IRB-HSR) committee and followed procedures with ethical standards.Categorical data are summarized by frequencies and percentages, and continuous scaled data are summarized by the median and interquartile range, and range of the empirical distribution.For the AEC related univariate analyses, the subject-specific AEC data represents the subject’s average of maximum annual AEC values over the study period, while for the IgE related analyses, the subject-specific IgE data represents the subject’s average of maximum annual IgE values over the study period.The relationship between AEC and lung function and the relationship between IgE and lung function was examined in two ways. In the first analysis, AEC/IgE served as the outcome variable, and lung function served as the stratification variable, where lung function was stratified into the following ordinal categories: normal, mildly impaired, moderately impaired, and severely impaired based on ppFEV1. The nonparametric Kruskal–Wallis test was used to compare the lung function stratified AEC/IgE empirical distributions. In the second analysis, the ordinal categories for lung function served as outcome variable, and AEC/IgE served as the predictor variable. Ordinal logistic regression (OLR) was used to examine if a higher level of lung function impairment is associated with higher levels of AEC/IgE. Outcome versus predictor association was quantified by the OLR proportional odds ratio test (POR), and proportion odds ratio represented relative difference between odds of having a higher level of lung function impairment between two hypothetical subjects: one with an AEC/IgE value equal to the 75th percentile of the AEC/IgE empirical distribution, and the other with an AEC/IgE value equal to the 25th percentile of the AEC/IgE empirical distribution.The relationship between AEC or IgE and the number of annual pulmonary exacerbations was examined in exactly the same way as the relationships between AEC and IgE lung function. A CF exacerbation was defined as a respiratory exacerbation event that required intravenous antibiotics and/or hospitalization. For both sets of analyses the number of pulmonary exacerbations was ordinally categorized as: 0, 1–2, and > 2 pulmonary exacerbation per year per person. For example, if a person had 3 exacerbations per year in any given year of the study period, he would be included in the analysis as > 2 pulmonary exacerbations.Mycobacteria spp., (B) Methicillin Resistant Staphylococcus Aureus (MRSA), (C) Pseudomonas aeruginosa, (D) Stenotrophomonas spp., (E) Achromobacter spp., (F) Aspergillus fumigatus and other Aspergillus spp., (G) Exophiala spp., (H) Rasamsonia spp., and (I) Scedosporium spp. Per microbiological organism, 2 sets of analyses were conducted. One set of analysis compared via the nonparametric two-sample Wilcoxon Rank Sum test the AEC/IgE empirical distributions of those subjects who had positive sputum tests for the microbiological organism to the AEC/IgE empirical distributions of those subjects who had negative sputum tests for the microbiological organism. The second set of univariate analyses was focused on determining via logistic regression (LR) if the subjects AEC/IgE levels could predict the presence of the microbiological organism. The LR odds ratio test compared the odds for testing positive for the microbiological organism between a hypothetical subject who has an AEC/IgE level equal to the 75th percentile of the AEC/IgE empirical distribution, relative to a hypothetical subject who has an AEC/IgE level equal to the 25th percentile of the AEC/IgE empirical distribution.AEC and IgE levels were compared between subjects who tested positive and subjects who test negative for: (A) Stenotrophomonas spp., Achromobacter spp), Aspergillus spp., or other fungi  and separately, a positive allergen panel  [Longitudinal data were analyzed using mixed effects models with a random effect of patient built in R using the lme4 and lmerTest packages. Two sets of secondary models were built that considered covariates age, gender, ppFEV1, and presence of Pseudomonas spp., MRSA, other bacteria ( panels) . AEC andFor all analyses, a two-sided p ≤ 0.05 decision rule was used as the null hypothesis rejection criterion.Pseudomonas aeruginosa of 70%, MRSA of 32.9%, and Mycobacteria spp. of 20.8% over the study period. Notably, when highest AEC per year were averaged over the study period 34 (45.9%) patients had AEC levels above 300 cells/μL. In our cohort, 18.9% of the patients had a maximum IgE level >180 IU/mL. Most of the patients in our sample were on inhaled corticosteroids and CFTR modulators . Greater AEC was not a significant predictor of disease severity . With respect to IgE, there was a detectable shift in the central location of distribution between the 4 different levels of lung function impairment (p = 0.049), with the highest median IgE value for the group of patients with moderate lung function impairment. However greater IgE was not a significant predictor of disease severity. .Pseudomonas spp., MRSA, other bacterial spp., Aspergillus spp, and other fungi (p < 0.001). Also in this model, increased age was correlated with a decline in ppFEV1 (p = 0.0396), while there was no statistically significant relationship between ppFEV1 and the other covariates . This negative association remained statistically significant even when adjusting for age, gender, presence of A linear mixed model showed demonstrated a statistically significant correlation between ppFEV1 decline and IgE increase (p < 0.001). This correlation remained statistically significant in a multivariate linear mixed model when after adjusting for age, gender, presence of Pseudomonas spp., MRSA, other bacterial spp., Aspergillus spp, and other fungi (p < 0.001). In this model, increasing increased age was also correlated with a decline in ppFEV1 decline (p = 0.04).  Fig. Pseudomonas spp., MRSA, other bacterial spp., Aspergillus spp, and other fungi, the effect of CF exacerbation was no longer significant (p = 0.4372). However, presence of a CF exacerbation correlated with ppFEV1 decrease (p = 0.0003). In addition, per each additional annual exacerbation, the annual variance in AEC increased (p < 0.001), an effect that remained significant after adjusting for age, gender, ppFEV1, presence of Pseudomonas spp., MRSA, other bacterial spp., Aspergillus spp., and other fungi (p = 0.0001). Logistic regression showed that as log AEC increases, the probability that the measurement was taken during a CF exacerbation increases (p = 0.0039) .A longitudinal model comparing AEC within and outside of exacerbation events showed that log AEC was lower outside exacerbations when compared to levels related to an exacerbation event p = 0.0035) .A linear mixed model showed that each additional annual exacerbation event increased on average the log IgE by 0.04. (p = 0.015) Fig. Aspergillus spp. (p = 0.0003) and decreasing ppFEV1 (p = 0.0110) as significant predictors of the number of CF exacerbations.The presence of a positive serum allergen panel further influenced this correlation. A linear mixed model showed an increase in log IgE by 0.707 for each additional annual exacerbation (p = 0.0044) and significantly higher log IgE in allergen positive patients (p < 0.001). While a Poisson mixed model explaining the number of annual CF exacerbations with just log IgE showed no effect of log IgE (p = 0.2263), the model including atopy showed that in the presence of atopy based on positive serum allergen panels, as log IgE increases the exacerbation events increase (p = 0.0222) Fig. Aspergillus, but there was a marginal upward shift in center location of the AEC empirical distribution due to presence of Exophiala or Rasamsonia or Scedosporium (p = 0.076). AEC was not a predictor of the presence of Aspergillus , nor was AEC a predictor of the presence of Exophiala or Rasamsonia or Scedosporium .Univariate analysis. Empirical distribution numerical summaries are provided in Additional file Aspergillus, and there was no shift in center location of the IgE empirical distribution due to presence of Exophiala or Rasamsonia or Scedosporium. IgE was not a predictor of the presence of Aspergillus , nor was IgE a predictor of the presence of Exophiala or Rasamsonia or Scedosporium .With respect to IgE, there was no shift in the central location of the IgE empirical distribution due to the presence of Pseudomonas, MRSA, Achromobacter or Stenotrophomanas, but there was a marginal upward shift in center location of the AEC empirical distribution due to presence of Mycobacteria (p = 0.099). AEC was not a predictor of the presence of Pseudomonas, MRSA, Achromobacter or Stenotrophomanas, or Mycobacteria (Table 5B). With respect to IgE, there was no shift in the central location of the AEC empirical distribution due to the presence of Pseudomonas, MRSA, Achromobacter or Stenotrophomanas, or Mycobacteria. IgE was not a predictor of the presence of Pseudomonas, MRSA, Achromobacter, Stenotrophomanas, or Mycobacteria .Prior to the introduction of highly effective CF modulator therapy, the CF Foundation patient registry report showed that at any given year, approximately 40% of patients had one or more exacerbations a year requiring hospitalization and/or intravenous antibiotics and approximately 10% of this group had 3 or more exacerbations a year. Each year, there is a slight change in the patient groups, some patients do not develop exacerbations annually but every other year or every 3 years and some patients suddenly develop more active disease, and more frequent yearly exacerbation. Following this cohort longitudinally allowed us to include in the analysis more patients with less  and more frequent exacerbations (more than 2) or more active disease, over a longer period of time. In addition, when a patient develops more than 2 exacerbations a year, our group initiates a frequent exacerbation protocol to identify possible new contributors to the change in phenotype which includes evaluation of ABPA/allergic or atopic phenotype based on serum IgE and AEC levels. Of note, our population has a high exacerbation rate, with 57.5% of our patients having 1–2 exacerbations per year and 24.5% of patients having 3 or more exacerbations per year. While this could be a result of our center’s geographical location it can also be a result of our study period (6 years), which allowed more incidences to be counted than otherwise i.e., even if a patient had only 1 exacerbation in the study period, they would be counted in the 1–2 Exacerbations per year cohort. In addition, our center monitors very closely the AEC and IgE levels with each exacerbation, which most likely led to an increased identification of elevated levels for these two measurements.As per our hypothesis, higher levels of AEC were associated with more frequent exacerbations, defined as > 2 pulmonary events that required hospitalization and/or IV antibiotics. Our study shows that CF exacerbations increase the AEC levels and corelate with higher AEC variability. The correlation between CF exacerbation and AEC may be explained by exposure to intravenous antibiotics, however the correlation between CF exacerbations and AEC variability is a new finding that is worth further exploration. Based on our study however we cannot draw a conclusion on a cause and effect. It is unclear if exacerbations cause increased T2 inflammation or T2 inflammation predisposes to increased exacerbations.When accounting for positive allergen panel positivity, as IgE increases, exacerbation events increase. In addition, each annual exacerbation is associated with an increase in IgE and this effect was significantly more pronounced in patients with atopy. This is a new finding and suggests that T2 inflammation may be partially responsible for increased exacerbations. If proven in multicenter, larger study, could lead to a change in CF management with increased use of ICS, and asthma immunological therapies. T2 inflammation leads to reduced expression of antimicrobial proteins, goblet cell hyperplasia and increased mucus production which subsequently can lead to increased bacterial burden, increased respiratory symptoms and subsequentially, increased pulmonary exacerbations .Pseudomonas spp., MRSA, other bacterial spp., Aspergillus spp, and other fungi. Our population is older, with a mean of 35 years, and also has a higher prevalence of Pseudomonas (70%), MRSA (32%), and Mycobacteria (21%) than previously reported in other population cohorts. Despite the high infection rates, only 23% of the patients had frequent (> 2) exacerbations/year at any time during the study period. Moreover, neither AEC nor IgE were statistically associated with any bacterial strain. We believe this indicates that the relationship between AEC and pulmonary exacerbations is independent of bacterial presence. While the exact nature of this relationship is unclear, research has shown that eosinophils participate in pulmonary pathology [Another novel finding of our study is that longitudinal analysis showed that ppFEV1 decline correlated with increased AEC and IgE even when adjusting for age, gender, presence of athology . This coExophiala spp, Rasamsonia spp, and Scedosporium spp. have been shown to cause pulmonary disease in CF patients [Aspergillus spp despite the high prevalence of Aspergillus in our population (69%). In the models with covariates, exacerbation rates did not correlate with bacterial colonization but did correlate with Aspergillus colonization suggesting that perhaps frequent antibiotic and steroids exposure increases the risk of Aspergillus colonization. This is an interesting finding as Aspergillus can generate a T2 response and has been associated with ABPA in the CF population [While bacteria were not correlated with either AEC or IgE, the E/R/S fungi were almost statistically-significantly correlated p = 0.076) with elevated AEC. We decided to evaluate these fungi despite their low sample size as patients , 16. The6 with elpulation  observatUVA is situated in Charlottesville, Virginia which is on the asthma belt. Most of our subjects come from rural Virginia areas and work on farms or own farmlands and frequently work with animals and are exposed to multiple allergens. Virginia is a hot-humid, forest-heavy state. Our center’s geographical location and humid weather exposes our patients to a large variety and concentration of pollen and mold allergens. We recognize that a weakness of this study is the utilization of self-reported or physician-diagnosed asthma, and it is possible that the true prevalence of asthma is smaller. To avoid possible bias when using asthma as a variable in the analysis, we chose not to explore this diagnosis. According to the Virginia Department of Health, the 2016 asthma prevalence in Virginia was 8.6%, compared to the national average asthma prevalence of 7.7%. Environmental exposures and/or asthma exacerbations could underlie both correlations between AEC and pulmonary exacerbations and E/R/S respectively. The geographical environment may skew our patient population towards a T2 phenotype. Our patient population is also older with a mean age at study time of 35 which could also be a confounder as there is an increase the risk of sensitization in our patient cohort with increasing age. We found age to be a statistically significant covariate in some models with covariates and, as expected, age correlated with ppFEV1 decline.Exophiala spp, Rasamsonia spp, and Scedosporium spp due to the low number of cases, which may impact findings.There are several limitations to this study and further research is required to better understand the implications of elevated peripheral absolute eosinophils counts in CF disease. We are limited by our study’s retrospective design, unique geographical distribution, and being a single center study with a limited number of patients. Given the retrospective limitations of the study, we could not account for oral steroid use during the study period. These are frequently prescribed in our patient population by outside providers. Highest average AECs were used for univariate analysis to account for this limitation. The AECs obtained during exacerbations were included in the analyses as we think they are a marker of T2 inflammation regardless of trigger . Another limitation of our study is the paucity of IgE data. In addition, as mentioned earlier, our study grouped together In conclusion, this study has shown that in CF patients, AEC correlated with frequent pulmonary exacerbations requiring hospitalizations and/or intravenous antibiotics, independent of bacterial airway colonization. Treatments that target eosinophilia, such as Mepolizumab , have positive effects in CF patients with T2 inflammation . With thAdditional file 1: Figure S1. AEC in exacerbations and during stable outpatient visits. AEC=Absolute Eosinophil Counts. Figure S2. Number of exacerbations and IgE levels in the presence and absence of positive allergen panels. Table S1. Patient characteristics based on AEC levels. Table S2. Patient characteristics based on IgE levels. Table S3. T2 Inflammation and Fungal presence. Table S4. T2 Inflammation and Bacterial presence.


Hydrogenophaga, Thauera, Planctomyces, Zoogloea, and Pseudomonas boosted from 0.25, 0.01, 0.00, 0.05, and 0.08% to 3.33, 3.43, 0.02, 6.28, and 2.69%, before and after raw sewage discharge, respectively. The gene abundance of pathogenic bacteria significantly increased after raw sewage discharge. For instance, the gene abundance of Vibrio, Helicobacter, Tuberculosis, and Staphylococcus augmented from 4055, 3797, 13,545, 33 reads at Site-1 to 23,556, 13,163, 19,887, 734 reads at Site-2, respectively. In addition, according to the redundancy analysis (RDA), the infectious pathogens were positively related to the environmental parameters, in which COD showed the highest positive correlation with Mycobacterium tuberculosis. Additionally, river self-purification may contribute to improving water quality and reducing pathogenicity. The outcomes of this study showed that direct discharge brought pathogens and changed microbial community structure of the river.Direct sewage discharge could cause copious numbers of serious and irreversible harm to the environment. This study investigated the impacts of treated and raw sewage on the river ecosystem. Through our analysis, sewage carried various nutrients into the river, leading to changes in the microbial community in the river and reducing the diversity and richness of bacteria. The relative abundances of  Moreover, a body of literature has confirmed the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wastewater4. SARS-CoV-2 possibly spreads through the wastewater treatment network5. When raw sewage contaminates the aquatic environment, these pathogens can be transmitted through the water to those who use the water for swimming, boating, and fishing, causing a range of potential risks6. However, there are many ways for untreated wastewater to enter rivers. When sewage treatment cannot treat too much wastewater or when it is raining heavily, raw sewage will be discharged directly into river channels, polluting the environment and making the water quality worse. Natural disasters, such as earthquakes and floods, also make raw sewage flow into rivers, transporting pathogens into them7. Once sewage is delivered to receiving waters, a series of environmental changes will occur. Over time, river dilution, storage in sediments, and the intrinsic characteristics of the microorganisms and other related processes may alter the destiny of the bacteria and pathogens of concern.With the massive worldwide population increase, water has been predicted to become one of the scarcest resources in the twenty-first century. Moreover, the UN’s World Water Development Report, 2017, said globally 80% of sewage (> 95% in developing countries) is directly released to the environment. Numerous questions have been raised about the ability of wastewater treatment programs to remove pathogens from wastewater, in which many waterborne diseases are associated with supposedly treated water supplies. Untreated wastewater can threaten the human and ecological systems by enriching pathogenic bacteria8, especially ammonia (NH4+), nitrate (NO3−), total phosphorus (TP), and chemical oxygen demand (COD). The increase of these contaminants could trigger the aquatic organism by causing eutrophication of the catchment9. David et al. found that the high NH4+ concentration negatively influenced the functional performance and taxonomic richness of the microbial community of 10 different WWTPs located across Switzerland10. Moreover, phosphorus content was positively correlated with the microbial biomass11 and occupied an important position among environmental factors that affect the microbial community of sediments13.The direct discharge of sanitary wastewater may cause environmental diversity changes. Sewage discharge without proper treatment significantly altered the concentrations of different organic and inorganic contaminants in the receiving water bodiesVibrio cholera, Staphylococcus aureus, Mycobacterium tuberculosis, and Helicobacter pylori, exist in aquatic environments. Vibrio cholerae, a well-known internationally quarantinable infectious pathogenic bacterium, could cause human enteric infection14. Vibrio cholera O1 is widely distributed in aquatic environments, namely rivers, ponds, sewage, and estuaries, in many developing countries such as Haiti15. Reidl et al. isolated Vibrio cholerae from estuarine and aquatic environments14. Staphylococcus aureus may cause local purulent infection, pneumonia, and colitis after enrichment in water16. Zieliński et al. found sewage treatment plants are a sizeable source of drug-resistant staphylococci harboring virulence genes17. Moreover, Mycobacterium tuberculosis normally enters the host via mucosal surfaces, usually through the lung after inhaling infectious droplets from an infected individual and occasionally via the intestine after ingesting infected material18. Velayati et al. isolated Mycobacterium tuberculosis from water samples collected in Tehran, Iran metropolitan area19. Many diseases, such as gastritis, peptic ulcer, and lymphoid proliferative gastric lymphoma, are caused by Helicobacter pylori infection20. West et al. reported the capabilities of Helicobacter pylori to survive in various buffers at room temperature over a range of physical variables, which means that Helicobacter pylori may survive in a natural aquatic environment21. Therefore, pathogenic bacteria in the water environment will have direct or indirect effects on human beings or animals. The study of their microbial community composition and influencing factors is urgent for future treatment measures.Several common infectious pathogens, such as 23. However, it is unclear whether the source of pathogenic bacteria in the river comes from sewage itself or whether it is caused by the environmental change after sewage discharge. Also, a deep insight into microbial and genetic responses to river self-purification needs further investigation. Thus, the main target of the present study is to fill in this knowledge gap by investigating the environmental parameters and the microbial community structures of different sites along the Daxin River, representing treated and raw sewage discharge points. To achieve this objective, four sample sites were selected before and after treated and untreated sewage discharge points. The microbial community composition was determined by high-throughput 16S rDNA amplicon sequencing. Also, the components and concentrations of pathogenic bacteria were identified by qPCR and forecasted by PICRUSt analysis. The concentration of environmental factors, including ammonia, nitrite, nitrate, phosphate, COD, and temperature of the sediment-overlying water, were analyzed. Moreover, redundancy analysis (RDA) was employed to explore the relationship between environmental factors and microbial community structure. The specific objectives of this study are to (1) provide more information about the water quality and microbial community structure before and after domestic wastewater pollution, (2) explore the composition of the pathogenic bacteria community in the Daxin River, and (3) evaluate the relationship of infectious pathogens and functional bacteria to different environmental factors.The lack of a thorough understanding of the survival and persistence of different microbial types in different conditions and environments is one of the major gaps in the knowledge of pathogenic microorganisms in wastewater. Previous studies focused on the abundance of pathogenic bacteria and the relationship between the contaminants and pathogenic bacteria in rivers contaminated by raw sewage dischargeJinan is located in northwest Shandong Province, China, near the confluence of two important water systems, the Yellow River and the Xiaoqing River. The Daxin River is a tributary of the Xiaoqing River. Sediment and sediment-overlying water samples were harvested from four different sites along the Daxin River. The four sites were designated, from upstream towards downstream of the river, as Site-1 , Site-2 , Site-3 , and Site-4 , representing the discharge points of treated and raw sewage 24. The sediment-overlying water temperature was measured by a centigrade thermometer at each site. The pH was measured using a pH meter  during water sampling. The content of total phosphorus (TP) was determined by the colorimetric method of vanadium-molybdenum phosphoric acid. The measurement of a COD was carried out by COD rapid measuring instrument .The concentrations of NO25. The qPCR  was used to detect the gene concentration of Vibrio cholerae, Staphylococcus aureus, Mycobacterium tuberculosis, and Helicobacter pylori. The primer sequences of the genes of Vibrio cholerae were GCCAAAATTGTGCGTATCAG and ATAATCTTGGGCAATCGCA. The primer sequences of the genes of Staphylococcus aureus were CTAATACTGAAAGTGAGAAACGTA and TCCTGCACAATCGTACTAAA. The primer sequences of the genes of Mycobacterium tuberculosis were CCTGCGAGCGTAGCGTCGG and CTCGTCCAGCGCCGCTTCGG. The primer sequence of the genes of Helicobacter pylori were GCCAATGGTAAATTAGTT and CTCCTTAATTGTTTTTAC. The qPCR reaction mixture (20 μL) included 2× SYBR Green PCR master mix (10 μL), forward primer (0.4 μL), reverse primer (0.4 μL), template DNA (1 μL), and dd H2O (8.2 μL). The primers used to identify the infectious pathogens were designed according to the conservative sequence of characteristic genes. The annealing temperature of qPCR was predicted using the software primer5.0 based on its “GC” ratio in the whole sequence26. The qPCR data were analyzed using Abs Quant/2-order derivatives of Max software27.The Power Soil™ DNA Isolation Kit (Takara Company) was used to extract the total DNA of the freeze-dried sediment samples28. The V4 amplicons were sequenced using primers 515F (GTGCCAGCMGCCGCGGTA) and 806R (GGACTACNVGGGTWTCTAAT) and the pair-ended method on the Illumina Miseq sequencing platform at Personal Biotechnology Co., Ltd.29. PICRUSt was used to predict the potential of a sample using 16S rDNA amplicon sequencing. KEGG Orthology (KO) was used to classify all genes that are homologous to a specific gene whose function is known to be in the same category16.The next-generation sequencing was applied to determine the microbial community structure based on the universal primer pair of 16S rDNAStaphylococcus aureus and Escherichia coli, were selected to investigate the effects of river water and sediment on microbial growth at different pollution levels. Unpolluted river water, Unpolluted sediment, polluted river water, polluted sediment, recovered river water, and recovered sediment samples were collected and made as culture media for the growth of the two bacteria, and the biomass-time curves of the bacteria were examined to determine and verify the dominated environmental media that promote bacterial growth in rivers. The water samples from Site-1, Site-2, and Site-4 were collected and filtered using 0.22 μm polyethersulfone membrane to make as unpolluted water culture medium, polluted water culture medium, and recovered water culture medium. The sediment samples from Site-1, Site-2, and Site-4 were centrifuged at 1000g for 5 min. The supernatant was decanted and the centrifuged sediment was retained. Then, 10 mL deionized water was added to the sediment, and the sediment was sonicated for 20 min to extract the nutrients from the sediment, followed by centrifugation at 1000g for 5 min. The supernatant was extracted and filtered using 0.22 μm polyethersulfone membrane to make as unpolluted sediment culture medium, polluted sediment culture medium, and recovered sediment culture medium. All culture media were sterilized by autoclaving at 121 °C for 15 min. The growth curves of both bacteria were examined using an automated microbiology growth curve analysis system . The volume of culture media was 1 mL, including 1% of bacterial seed solution . The temperature was set at 30 °C and the value of OD600 nm was recorded every 60 min.To characterize the environmental media affecting microbial growth in Daxin River ecosystems, typical Gram-positive and negative bacteria, 30. CANOCO version 4.5 software was used for RDA to evaluate the relationship between the environmental parameters and microbes.The community richness was estimated by the Chao1 and community diversity was estimated by Simpson and Shannon indices using the MOTHUR software4+-N concentrations significantly increased from 47.3 and 25.6 mg/L to 74.3 and 73.3 mg/L, respectively, after the raw sewage was directly discharged into the river. Likewise, NO3–N and TP concentrations slightly increased from 0.2 and 1.6 mg/L at Site-1 to 1.2 and 5.7 mg/L at Site-2, respectively. However, due to river self-purification that occurred between Site-2 and Site-3, all sediment-overlying water quality parameters returned to their average level before the discharge of raw sewage, except for COD which witnessed a slight decrease from 74.2 to 62.8 mg/L at Site-3. In contrast, there were relatively small changes after the discharge of sewage treatment plant effluent to the river. COD slightly decreased from 62.8 to 52.3 mg/L, while NO3–N increased from 0.23 to 12.2 mg/L at Site-3 and 4, respectively.The sediment-overlying water quality parameters of the four sites were summarized in Table 31. As shown in Table The microbial community structure was determined by high-throughput 16S rDNA amplicon sequencing. The total obtained operational taxonomy units (OTUs) were 28,427, 28,354, 32,810, and 39,105 for the four sites, respectively. The Chao1, Simpson, and Shannon indexes were calculated to determine the richness and diversity of the microbial communityProteobacteria increased from 11.8% at Site-1 to 88.2% at Site-2 and then gradually decreased to 62.54% at Site-3 and 38.2% at Site-4. Conversely, relative abundance of Chloroflexi decreased from 7.18% (Site-1) to 0.53% (Site-2) and then gradually recovered to 5.52% (Site-3) and then 9.58% (Site-4); Spirochaetae decreased from 14.67% (Site-1) to 0.3% (Site-2) and then gradually recovered to 4.68% (Site-4); Euryarchaeota decreased from 31.37% (Site-1) to 0.07% (Site-2), however, they only recovered to 0.91% (Site-4). Additionally, Actinobacteria, Acidobacteria, Planctomycetes, and Caldiserica were reduced slightly to 0.41, 0.32, 0.05, 0.01% at site-2, respectively, and gradually recovered to 3.94, 2.02, 0.94, 0.15% at site-4, respectively. The ten dominant genera were shown in Fig. Treponema, which can convert NO2− to NO3−, had the highest abundance percentage of 10.42% at Site-1. Two denitrifying bacteria were enriched at Site-2, i.e., Hydrogenophaga (3.33%) and Thauera (0.86%). Thauera, a denitrifying related bacteria producing N2 using NO2− or NO3− as the electron donor32, was also enriched at Site-4. Azoarcus that performs nitrogen fixation33 appeared at Site-2 then kept growing in Site-3 and Site-4. The Zoogloea and Pseudomonas genera whose main function is to degrade organic matters increased sharply at Site-2. The Pseudomonas genus, responsible for the removal of phosphorus, was also dominant in Site-2 coupled with Acinetobacter. Their enrichment may be due to the high phosphorus content in the raw sewage, as evidenced by the highest TP content at Site-2 , anammox bacteria, organic degrading bacteria (ODB), and phosphorus-accumulating bacteria (PAOs) as shown in Table Hydrogenophaga at Site-2 (3.333%) was more than 13-fold of that at Site-1 (0.253%); however, it suddenly decreased at Site-3 and Site-4. Azoarcus was absent at Site-1 and appeared at Site-2 (0.005%) then continued to grow at Site-3 (0.194%) and Site-4 (0.244%). Thauera also showed an increasing trend from Site-1 to Site-4. Anammox bacteria Planctomyces were also detected at the genus level, and its highest abundance was 0.59% at Site-3. Organic degrading bacteria Zoogloea increased significantly to 6.28% at Site-2 and decreased to 0.093% and 0.497% at Site-3 and Site-4, respectively. PAOs bacteria include Pseudomonas, Arthobacter, Nocardia, Beyerinkia, Ozotobacter, Aeromonas, Microlunatus, and Rhodocyclus25. Only one PAOs genus was detected in all sites. The abundance of PAOs was much higher at Site-2 than that at Site-1, with a relative abundance of 2.69% and 0.08%, respectively. The relative abundance of all the above-mentioned functional bacteria at Site-2 is higher than those at Site-1, while only four bacteria related to COD and nitrate removal showed a continuous increase between Site-3 and Site-4.Fifty-nine phyla were detected from the four sites, and ten predominant phyla were shown in Fig. 34. Figure Vibrio cholera pathogenic cycle process, whose gene amount increased from 4055 to 23,556 reads. The gene abundance of Helicobacter pylori infection increased from 3797 to 13,163 reads. The gene abundance of Tuberculosis also increased from 13,545 to 19,887 reads. The four kinds of pathogenic genes had the most remarkable variation among all the genes related to infectious diseases between Site-1 and Site-2. On the other hand, there is no remarkable big gap between the gene abundance of samples at Site-3 and Site-4. The values of these four infectious disease abundances were verified by qPCR, as shown in Fig. Staphylococcus aureus at Site-2 far exceeded that of Site-1. The abundance of these four infectious diseases decreased at Site-4, compared to Site-3.Pathogenic bacteria in water affect human and animal health. Pathogens could live in untreated wastewater for longer periods and spread further through water bodies2−, NH4+, NO3−, TP in Table 4+ concentrations than other locations. Meanwhile, TP and NH4+ got a strong positive correlation with each other among all the environmental factors and they contributed to a large proportion in affecting the abundance of Mycobacterium tuberculosis  Fig. . The datStaphylococcus aureus, and Escherichia coli, were selected to examine their growth potential in different media. Figure Staphylococcus aureus (a) and Escherichia coli (b) in media from different sampling sites. Under the exposure of unpolluted water in Site-1, the bacterial densities of Staphylococcus aureus and Escherichia coli were almost unchanged and the value of OD600 nm was maintained around 0.073. The unpolluted sediment still contained various nutrients, which slightly promoted the growth of Staphylococcus aureus and Escherichia coli, with OD600 nm values reaching 0.079 and 0.078 at 1000 min, respectively. The polluted river water and sediment collected at Site-2, however, allowed the rapid growth of Staphylococcus aureus and Escherichia coli, with the OD600 nm values reaching 0.120 and 0.117 at 1000 min, respectively. While the recovered river water obtained at Site-4 also promoted the growth of these two bacteria slightly, with both values of OD600 nm reaching about 0.078 at the 1000 min. Compare with the recovered river water, the recovered sediment promotion was even more effective, with values of OD600 nm reaching 0.082 and 0.084 at 1000 min. These results suggest that the polluted river water and the sediment contain nutrients to support the growth of Staphylococcus aureus and Escherichia coli at ambient temperature (30 °C).This survey suggested that pathogenic bacteria could survive and multiply after sewage discharge. To investigate and verify the effect of various aquatic media on the growth of microbes, typical Gram-positive and negative bacteria, Staphylococcus aureus and Escherichia coli were tested respectively, as shown in Fig. Staphylococcus aureus and Escherichia coli became more effective as the mass addition of sediment increased.To further verify the effect of polluted water and sediment in the river on pathogenic bacteria, growth experiments on two typical strains were conducted with different polluted river concentrations and different sediment mass addition. First, culture media containing different volumes of contaminated river water was made up to 1 mL with sterile water. Under the exposure of different concentrations of polluted water, the growth curves of Proteobacteria, witnessed a noticeable growth after raw wastewater flowed into the river, as shown in Fig. Proteobacteria, Bacteroidetes, and Firmicutes were the most abundant phyla in all samples. Proteobacteria include various pathogens, such as Escherichia, Salmonella, Vibrio, and Helicobacter, and many bacterial species responsible for nitrogen transformation and had a positive correlation with NH4+ concentration35. The high relative abundance of Proteobacteria at Site-2 could be due to the increase in NH4+-N concentration from the direct discharge of sewage to the river; the high ammonia provided an appropriate living environment for Proteobacteria growth36. In a suitable environment, Proteobacteria competes with other native microorganisms and reduces microbial diversity38. With the gradual decrease of NH4+-N concentration at Site-3 and Site-4, the abundance of Proteobacteria gradually declined to 62.5 and 38.2%. Table Pseudomonas, which can remove low concentrations of TP from wastewater39. The discharge of raw sewage between Site-1 and Site-2 increased COD and nitrate contents, which provided sustainable and effective electron donors for denitrification, promoting the growth of denitrifying bacteria, such as Hydrogenophaga and Thauera. Zoogloea has been reported with high versatile metabolism capabilities such as nitrogen fixation, secretion of extracellular polymeric substance (EPS), and COD removal40. The increase of ammonia nitrogen content at Site-2 may be related to the high Zoogloea abundance at this site. The contents of Zoogloea increased at Site-2 after the raw wastewater surged in and then decreased in the following two downstream sites. RDA results suggested that functional bacteria, such as Zooloea, Pseudomonas, and Hydrogenophaga, were closely related to COD, NH4+, and TP. The DNB, which can restore NO2−, was enriched sharply at Site-2. The possible reason may be that NO2−, and NH4+ provide nutrition for DNB41.According to our analysis, the raw sewage altered the environmental conditions of the river, caused changes in the microbial community, and eventually led to a decrease in species richness and diversity. It may be due to the discharged treated sewage, containing high levels of nutrients, boosted the abundance of bacteria that thrive in eutrophic environments, and inhibited the growth of other bacteria. As pollutant concentrations decreased and water quality improved, the species richness increased again during the process from Site-3 to Site-4. Moreover, the abundance of pathogens, which belong to 43. This paper revealed that raw sewage remarkably contributed to the enrichment of pathogens in sediment samples of the river. From the results of PICRUSt prediction, the gene contents of four infectious diseases indeed increased after raw wastewater was discharged into the river, while most gene contents decreased after the discharge of treated wastewater. Further analysis proved that the PICRUSt prediction was in accord with the qPCR results, similar to a study conducted by Langille et al.44. In general, the discharge of raw sewage into rivers changed the water quality of rivers and caused the growth of pathogenic bacteria in these rivers. The abundance of pathogenic genes of Site-2 showed a significant increase, which was higher than the other three sites. With the purification of the river, the water quality gradually changed and the abundance of pathogenic genes decreased in Site-3. When the treated wastewater was discharged into the river, the pathogenic genes of site-4 did not change dramatically. Moreover, in the case of sediments being disturbed, it is likely that microorganisms, including pathogens, would remigrate into the sediment-overlying water column. Medema et al. reported that protozoa can settle from the water column into the sediment and, due to their size, remain undisturbed for long periods45. Thus, re-suspension events increase the potential risk potential to human health for those who are involved in activities in the aquatic environment. A suitable temperature is also an essential factor for the growth of pathogenic bacteria. When the pathogen is sown in pasteurized sewage, it can be infectious for up to 7 days, in deionized water at 25 °C for 22 days, and in these media for up to 4 weeks or more at lower temperatures (< 4 °C)34. Vibrio pathogens could be easy to remain active during treatment in rural sewage treatment plants and appear to be more abundant over a wide temperature range of 17.1 to 27.2 °C46. Furthermore, it was reported that the relatively high temperature in the range of 5–30 °C was beneficial to the growth of Vibrio cholera47. These demonstrate the temperature tolerance of these bacteria in water body. Therefore, the suitable temperature in Site-2 also contributed to the survival and maintenance of the pathogens. In addition, with the change of sediment-overlying water quality, mostly COD, in the river from Site-2 to Site-3, it was more suitable for Mycobacterium tuberculosis to be enriched than other bacteria.The discharge of pollutants from industrial processes causes adverse environmental impacts on aquatic ecosystems, such as eutrophication of water bodies, the introduction of pathogens, re-assembling of the microbial community structure in rivers48. Suzuki et al. found E. coli grew in river water when sterilized sediment was supplemented49. Based on our results, pathogens in the Daxin River could come from the direct discharge of sewage and also could be enriched in the polluted aquatic environment. Sewage carried pathogenic bacteria and amounts of nutrients, strongly impacting the original river aquatic ecosystem. The polluted river water and sediment also contributed to the growth of pathogenic bacteria carried by the sewage and originally present in the river, which has been proved in the incubation experiments.Through the microbial community structure analysis, the microbial diversity at Site-2 decreased while the abundance of pathogenic bacteria increased. Here, the relationship of microbes and environmental factors was studied through incubation experiments for the growth potential of typical bacteria. Cui et al. found that the enteric pathogens in the urban rivers were likely to have originated from domestic sewage4+, and TP gradually decreased from Site-2 to Site-4. The reason behind the improvement in water quality from site-2 to Site-3 is mostly due to river self-purification, which may entail involve physical, chemical, or biological processes that contribute to the removal of pollutants from the river50. Furthermore, Site-4 has superior water quality over Site-2 and 3. This might be because the treated wastewater released from STP prior to Site-4 had fewer pollutants than Site-3, which in turn diluted the water and enhanced its quality. With the pollutant concentrations decreased, the abundance of genes related to Vibrio cholerae, Staphylococcus aureus, and Helicobacter pylori decreased and the diversity and richness of microbial community structure also recovered over time. The response of the ecosystem to chemically-driven environmental changes is modulated by physical and biological phenomena. Self-purification could keep the maintenance or restoration of river ecology and influencing factors may include seasonal fluctuations, the microbiological community present42, and temperature51. The biological processes in river ecosystem are mainly carried out by endogenous and exogenous microorganisms that can remove organic and inorganic pollutants in contaminated water bodies, essential for maintaining a stable ecosystem dynamics state, in terms of function and structure52. Another concern is that microbes could be shielded by physical embedding in organic matter, suspended particles, and occlusion of a biofilm, making them less susceptible to the inactivation action of disinfectants50. Most pathogens, which came from raw sewage, could also be absorbed by animals, plants, and sediments in the river. Therefore, it is urgent to further focus on the migration and transformation mechanism of pathogenic bacteria in sediments. Although the microbial structure was slightly restored and the abundance of most pathogenic bacteria was also slightly reduced through river self-purification, direct discharge of raw sewage caused severe and short-term irreversible damage to the river environment.After the raw sewage entered the river, the self-purification influenced the river's environmental conditions and then affected the further migration and deposition of pathogenic bacteria. The content of COD, NH53. This paper brings to the fore the need for comprehensive research into the movement and behavior of these microorganisms in river sediment after raw sewage is contaminated. And besides, with the evidence of increased pathogen abundance, we are here to demonstrate and highlight the risks of direct sewage discharge. Wastewater as a secondary source of transmission is not given full attention, especially in third-world countries. If wastewaters act as the source of transmission, it will be hard to break the chain of pathogen transmission in the third world countries and the consequence will be faced by developed nations as well. There is a common practice to discharge wastewater directly into rivers, canals, and lakes without any treatment in third-world countries. The wastewater carried pathogens can go to a big population of the world and create population crises in the world. Therefore, measures should be taken as soon as possible in third-world countries to break the chain of pathogen transmission. Strict surveillance of wastewater treatment is suggested to stop the spread of pathogens in the human community.This study contributes to an improved understanding of the effects of sewage discharge on the changes of microbial communities and the abundance of pathogenic bacteria in river sediments. Despite water and sewage treatment technologies continue to achieve rapid progress in recent years, waterborne pollution remains a major threat to public health worldwideHere, the effect of raw sewage discharge on the river system was studied. Direct discharge brought high concentrations of pollutants to the river, especially COD and nitrogen fractions. The deterioration of water quality had a serious impact on the surface mud microbial community of the river, and microbial diversity and abundance were reduced. The relative abundance of nitrogen metabolizing functional microorganisms and pathogenic bacteria increased after the direct discharge of sewage. Pathogens in the Daxin River could come from the direct discharge of sewage and also could be enriched in the polluted aquatic environment. As the river flowed, the water quality and microbial community structure of the river gradually recovered, and the abundance of pathogenic bacteria continued to decline, showing the self-purification ability of rivers. Although self-purification of rivers can restore a certain microbial diversity, the sewage direct discharge still causes irreversible effects, and river ecosystems cannot be restored to their original state. In conclusion, direct discharge of wastewater should be forbidden with regard to the negative impact on entering the ecosystem, especially on the lives that depend on the ecosystem for sustenance.