Perfluoroalkyl acids(PFAAs) are highly persistent chemicals thatare ubiquitously found in the environment. The atmospheric degradationof precursor compounds has been identified as a source of PFAAs andmight be an important pathway for contamination. Lake Vätternis one of Sweden’s largest lakes and is an important sourcefor drinking water. In addition to contamination via atmospheric deposition,the lake is subject to several potential contamination sources viasurface water inflow. The relevance of different sources is not wellunderstood. A mass balance of selected PFAAs was assembled based onmeasured concentrations in atmospheric deposition, surface water fromstreams that constitute the main inflow and outflow, and surface waterin the lake. The largest input was seen for trifluoroacetic acid (150kg/year), perfluoropropanoic acid (1.6 kg/year), perfluorobutanoicacid (4.0 kg/year), and perfluoro-octanoic acid (1.5 kg/year). Bothatmospheric deposition and surface water inflow was found to be importantinput pathways. There was a positive correlation between the inputof most perfluoroalkyl carboxylic acids via atmospheric depositionand global radiation and between the input via surface water inflowand catchment area. These findings highlight the importance of atmosphericoxidation of volatile precursor compounds for contamination in surfacewaters. Atmospheric oxidation of volatileprecursors is a majorsource of perfluoroalkyl carboxylic acids to a freshwater lake viaatmospheric deposition. Their amphiphilic properties makePFAAs useful in a wide range of applications. The use of PFAA-containingaqueous film-forming foam (AFFF) for firefighting training has beenidentified as a major source of groundwater contamination.6 The much less-studiedultrashort-chain PFAAs are defined as the PFCAs and PFSAs with a chainlength of 2–3 and 1–3 carbon atoms, respectively. Theseinclude trifluoroacetic acid (TFA), perfluoropropanoic acid (PFPrA),trifluoromethane sulfonic acid (TFMS), perfluoroethane sulfonic acid(PFEtS), and perfluoropropane sulfonic acid (PFPrS).PFAAs includeperfluoroalkyl carboxylic acids (PFCAs) and perfluoroalkylsulfonic acids (PFSAs), which are divided into short- and long-chaincompounds depending on the length of their carbon backbone. Long-chainPFCAs and PFSAs are defined as those containing at least eight andsix carbon atoms or more, respectively, and have a greater potentialfor bioaccumulation.7 and has been identifiedas a major source of TFA to the environment.8 A recent study on ice cores shows an increased deposition of TFA,PFPrA, and perfluorobutanoic acid (PFBA) since 19909 and that the environmental levels of TFA are expected toincrease in the future as a result of a shift from HFCs toward hydrofluoroolefins(HFOs).10Increasedattention to TFA was paid in the late 90s after the implementationof the Montreal Protocol in 1989, which aimed at phasing out ozone-depletingchlorofluorocarbons that were used as refrigerants. This resultedin the introduction of hydrofluorocarbons (HFCs) and hydrochlorofluorocarbons(HCFCs). The atmospheric degradation of HFCs and HCFCs results inthe formation of TFA11 at firefighting training sites, and in landfill leachate.12 The sources and environmental occurrence ofultrashort-chain PFAAs are not as fully documented as those of theirlonger chain homologs. While the occurrence of TFA has been frequentlydocumented in surface waters, only a few studies have included otherultrashort-chain PFAAs. Ultrashort-chain PFAAs have been reportedin surface water samples collected in connection to potential pointsources such as landfills, military training sites, and waste managementfacilities.13 PFPrA has been reported in atmosphericprecipitation14 and has been found in Arcticice cores.9 TFMS was recently reportedin the environment for the first time15 and also in surface water and groundwater far away from primaryenvironmental emission points.16 PFEtSand PFPrS have been reported in municipal and industrial wastewater.17 The potential sources and the environmentalfate of these substances are not yet well understood.TFA is a substance of multiplesources, and high concentrationshave been observed downstream a chemical plant,19 There are limited data availableon human exposure to ultrashort-chain PFAAs, but TFA was recentlydetected in human blood.20 The high polaritymakes ultrashort-chain PFAAs mobile in the environment, and accumulationin aquatic bodies has been observed.22 Their highpersistence, together with their mobility in the environment, makesthem a potential threat to drinking water, and it may be challengingand costly to remove contamination once adverse effects have beenidentified. Efforts have been made to remove PFAAs from drinking waterdue to the widespread contamination of groundwater; however, the effectivenessof commonly used technologies, such as granular activated carbon,decreases with shorter carbon chain length, and the smallest and mostpolar molecules are not removed at all.23Ultrashort-chainPFAAs are highly polar, and the potential forbioaccumulation in biota is low. However, accumulation in plants hasbeen shown.24 Field monitoring shouldthus aim for assessing less-studied chemicals in defined environmentalsystems to facilitate increased environmental understanding and decision-making.The present study includes input and output calculations of ultrashort-,short-, and long-chain PFAAs for one fresh water lake in Sweden  that is a drinkingwater source for almost 300,000 people and is subject to anthropogenicpressures such as wastewater discharges, landfill leachates, and agriculturaland industrial activity. In addition, two airports are located inthe catchment area, with elevated concentrations of PFAAs in bothgroundwater and surface water as a result.25 Occurrence of PFAAs in fish from the lake has previously been reported.26 The relative contribution of PFAA input fromdifferent sources, including local sources and atmospheric deposition,is not yet fully understood, and appropriate management strategiesof this drinking water reservoir is lacking. In the field of PFAAs,most studies have focused on short- and long-chain PFAAs. While theenvironmental occurrence of TFA has been well documented in the past,there is little published data on other ultrashort-chain PFAAs, andthe relevance of different sources has not been investigated, includingfor TFA.Thereis a need to understand the occurrence and mass flows ofultrashort-chain PFAAs to assess the environmental hazard and potentialfor human health effects. The environmental fate and behavior of pollutantscan be better understood by multimedia mass-balance models takingsources, emissions, mass transport, and chemical characteristics intoconsideration.The aim of the present study was to assess the relevanceof localand diffuse sources of PFAAs to a fresh water lake. A mass balanceassessment was performed for 14 PFAAs, including the ultrashort-chainPFAAs TFA, PFPrA, and TFMS based on measured inputs , outputs (surface water outflow), and theinventory of PFAAs in the lake. The correlation between the inputfrom atmospheric deposition and global radiation was examined to elucidatethe relevance of the atmospheric degradation of volatile precursorsas a pathway for PFAAs to the lake.Supplementary Information.Detailed information about chemicalsand reagents is provided in the n = 12) were collected on an island in Lake Vätternover 1 month of sampling time from July 2018 to June 2019. Open samplingcontainers were used to collect both wet and dry deposition. Depositionsamples (n = 9) were collected using polyethylenefunnels  connected to polyethylene containers. Snowsamples (n = 3) were collected in polypropylene boxesfrom December 2018 to February 2019. Surface water samples (grab samples)were collected from 19 streams (n = 76) at four occasionsfrom March to December 2019, representing four different seasons.Of the 19 streams, 17 streams were chosen as they represent the relevantsurface waters that discharge into Lake Vättern: one streamrepresents the main outflow, and another was included as it passesnear an airport with known PFAA contamination. The latter was notincluded in the mass balance as it discharges into another stream that discharges into Lake Vättern.The streams included in the study are subject to different anthropogenicpressures including sewage treatment plants, landfills, industries,firefighting training sites, and agriculture as well as stormwaterfrom polluted areas (some with known PFAA pollution). Reference samples(n = 4) were collected at four occasions from Marchto December 2019 from a stream upstream from the study area and downstreamfrom a lake that is not a recipient of any known local contamination.Effluent water samples were collected from a sewage treatment plant (n =4) and a paper mill (n = 4) that discharges directlyinto Lake Vättern at four occasions from March to December2019. Other sewage treatment plants and industries discharge intostreams, which are included in the monitoring. Surface water samples(grab samples) were collected from two locations in Lake Vätternduring spring and summer 2019. Samples were collected at 0.5 m depthin April when the water column is mixed (n = 2) andat 0.5 and 30 m depths in July or August when the lake is stratified(n = 4). Information about the lake mixing regimewas obtained by oral communication with the water management associationVätternvårdsförbundet. The sampling locationsare shown in Figure S1, and the potentialcontamination sources, catchment areas, and flow rates of respectivestreams are listed in Table S1.Bulk atmosphericdeposition samples, surface water from inflowing streams (500 mL), surface waterfrom Lake Vättern (1000 mL), and effluent from the sewage treatmentplant and the paper mill samples (500 mL) were extracted by weak anionexchange solid-phase extraction following the ISO25101 method withsome modifications (details are provided in the SI).Surface water samples and effluent from the sewage treatmentplant and the paper mill were filtered with glass microfiber filters(1.2 μm) prior to extraction to prevent clogging of the solid-phaseextraction sorbent. The filters were extracted three times with methanolby ultrasonication for 30 min followed by centrifugation at 71001–C4 PFAAs after some modifications for improvedseparation of ultrashort-chain PFAAs, including TFMS.27 An SFC Torus DIOL column   maintainedat 50 °C was used to achieve chromatographic separation. Separationand quantification of C5–C12 PFAAs wereperformed using ultraperformance liquid chromatography (UPLC) MS/MS operated in negative electrospray ionizationmode. A UPLC BEH C18 column   maintained at50 °C was used to achieve chromatographic separation. Detailedinformation about the SFC, UPLC, and source parameters is providedin the SI. At least two MS/MS transitionswere monitored for each target analyte except for TFA, PFPrA, PFBA,and PFPeA, where only one transition was monitored. MRM transitionsfor all target analytes are provided in Table S2.A novel analytical technique using supercriticalfluid chromatography(SFC) coupled with tandem mass spectrometry (MS/MS)  operated in negative electrospray ionizationmode was used for the separation and quantification of Cn = 11) at a concentration of 1 ng per 250 mL sample.The RSD was in the range of 11–17%. Extraction efficiencieswere assessed based on the peak area of native standards spiked totest samples (n = 11) after subtraction of the backgroundconcentrations in the samples. The extraction efficiency was in therange of 58–123%. Mass-labeled recovery standards were addedto the sample extracts prior to injection to monitor the recoveryof the method. The methanol used was checked for contamination, andprocedural blanks were included in each batch of samples. None ofthe target analytes were observed in the methanol. Limits of detection(LOD) were calculated as the average concentration in repeated proceduralblank extractions plus three times the standard deviation for samplesof atmospheric deposition (n = 8) and for samplesof surface water, lake water, and effluents (n =8). For those analytes that were not observed in procedural blanks,the LOD was set as the lowest calibration point with a signal-to-noiseratio of at least 3. Field blanks for surface water (n = 4) and rain (n = 2) were included to ensure thatno contamination occurred during sampling. The field blanks were samplingcontainers with ultrapure water that were opened during sampling,closed, and transported back to the laboratory and treated in thesame way as the samples. None of the target analytes were observedin the field blanks. Detailed information about repeatability, extractionefficiencies, and LODs are provided in Table S3.Isotope dilutionwas used for quantification using mass-labeled internal standardsthat were added prior to extraction. Corresponding mass-labeled internalstandards were available for most target analytes, including for TFA.Mass-labeled PFBA and PFBS were used for quantification of PFPrA andTFMS, respectively. The repeatability of the method was evaluatedbased on the relative standard deviation (RSD) of fortified samples were included in the statistical analysisand subsequently reported. Spearman rank correlations between thePFAA input via atmospheric deposition and global radiation and betweenthe PFAA input via inflowing surface water and catchment area werecalculated.Nstreams (kg/year), was calculated based on themedian concentration in the surface water (kg/m3) and theflow rate per year (m3/year). Low-bound estimates (LBE)and high-bound estimates (HBE) were calculated based on the lowestand highest concentrations measured, respectively. The input fromsurface water inflow was calculated according toiC is thePFAA concentration (kg/m3) in stream i and iQ is the flow rate per year (m3/year) of stream i. The flow rate per yearwas retrieved from a database created by the Swedish Meteorologicaland Hydrological Institute and the Swedish Agency for Marine and WaterManagement and is based on measured data (n = 3)and model calculations using the Hydrological Predictions for theEnvironment (HYPE) model (n = 12) (Table S1). Data was not available for two streams, and theflow rate per year was calculated based on flow rate measurementsat four occasions during March to December 2019 and calibrated bylinear calibration against the modeled flow rate per year from anadjacent catchment area.The input from surface water inflow, Ndeposition (kg/year), was calculated based onthe measured concentration in atmospheric deposition (kg/m3) in monthly samples and the amount of atmospheric deposition overLake Vättern per month (m3/month). The input fromatmospheric deposition was calculated according toiC is thePFAA concentration (kg/m3) in atmospheric deposition collectedduring month i and iQ is the amount of atmospheric deposition (m3) over LakeVättern during month i. The amount of atmosphericdeposition is based on measurements by the Swedish Meteorologicaland Hydrological Institute at a meteorological station on the sameisland as the precipitation samples were collected (Table S4). The input from atmospheric deposition rests onthe assumption that the amount of atmospheric deposition is representativefor the entire surface area of Lake Vättern.The inputfrom atmospheric deposition, Neffluent (kg/year), was calculatedbased on the median concentration in the effluent (kg/m3) and the flow rate per year (m3/year). The LBE and HBEwere calculated based on the lowest and highest concentrations measured,respectively.iC is thePFAA concentration (kg/m3) in effluent i and iQ is the flow rate per year (m3/year) of effluent i.The inputfrom effluents from the sewage treatment plant and thepaper mill, Noutflow (kg/year), wascalculated based on the median concentration (kg/m3) inthe stream that represents the main outflow and the flow rate peryear (m3/year). LBE and HBE were calculated based on thelowest and highest concentrations measured, respectively. The outputfrom surface water outflow was calculated according toC is the PFAA concentration(kg/m3) in the outflowing stream and Q is the flow rate per year (m3/year). The flow rate peryear was collected from a database created by the Swedish Meteorologicaland Hydrological Institute and the Swedish Agency for Marine and WaterManagement and is based on measured data.The output from surfacewater outflow, Table S4. Transformation in water was estimated to account for lessthan 0.5% of the output for PFHxA, PFOA, PFDA, and PFOS in the BalticSea.5 Sediment burial is not thought tobe relevant for ultrashort-chain and short-chain PFAAs but might berelevant for long-chain PFAAs. Sediment burial has been estimatedto account for 1.5% (PFHxA), 3.6% (PFOA), 26–32% (PFDA), and9.5% (PFOS) of the output in the Baltic Sea.5Other output pathwayssuch as transformation of PFAAs in water, volatilization, evaporation,groundwater recharge, and sediment burial were not considered. Annualevaporation from the lake is provided in Mwater (kg), was calculated based on the averageconcentration in the water basin (kg/m3) and the volumeof water in the water basin (m3). The concentration inthe water basin was calculated according toC is the PFAA concentration(kg/m3) in the water basin and V is thevolume of water in the water basin (m3).The inventory in Lake Vättern, Tables S5–S15. TFA was the mostabundant PFAA in both atmospheric deposition and in surface waterand was detected in all atmospheric deposition samples analyzed atconcentrations ranging from 18 ng/L in January 2019 to 300 ng/L inApril 2019. The monthly deposition flux of TFA was in the range from0.30 μg/m2 in January 2019 to 16 μg/m2 in July 2018. The annual deposition flux of TFA, calculated as thesum of the monthly deposition fluxes, was 52 μg/m2. This is approximately four times lower than the annual depositionflux of TFA reported in Germany in 2018.28 PFPrA was the third most abundant PFAA in atmospheric deposition(after PFBA) and was detected in all atmospheric deposition samplesanalyzed at concentrations ranging from 0.92 ng/L in December 2019to 3.7 ng/L in April 2019. The monthly deposition flux of PFPrA wasin the range from 0.02 μg/m2 in January 2019 to 0.26μg/m2 in July 2018. The annual deposition flux ofPFPrA was 0.78 μg/m2. An earlier study reported annualfluxes of 20 PFAAs, including PFPrA, in two locations in Japan andin the United States during 2006–2007 and 2007–2008.14 PFPrA was the only ultrashort-chain PFAA includedand was the most dominant compound in samples from both countries.Among the ultrashort-chain PFSAs, only TFMS was detected in two atmosphericdeposition samples in the present study in the concentration range0.13–0.15 ng/L. The deposition flux of TFMS was 0.007–0.01μg/m2 . To the best of our knowledge, the presence of TFMS in atmosphericdeposition samples is reported here for the first time. PFAAs in precipitationsamples have been documented in samples from remote and rural locations,32 but only a limited number ofstudies have reported the occurrence of ultrashort-chain PFAAs.Detailed information about the PFAA concentrations is providedin n = 79) at concentrations ranging from 30 ng/L to820 ng/L. The measured TFA concentration in most of the samples wasin the same range as previously reported in rivers in China34 but below the concentrations recently reported in rivers in Germany.35 PFPrA, TFMS, PFEtS, and PFPrS were detected in 73, 91, 42, and 38%of the samples, respectively, at concentrations in the range 0.60–2.9ng/L (PFPrA), 0.11–15 ng/L (TFMS), 0.24–0.62 ng/L (PFEtS),and 0.44–3.5 ng/L (PFPrS).In surface water from streams, TFA was detected in 91% of the samples in surface water from 2017, suggestinga buildup in concentration due to the annual input from atmosphericsources. To our knowledge, there are no previous studies reportingconcentrations of TFMS in surface water in lakes. Other studies havereported ultrashort-chain PFAAs in other matrices such as wastewater,17 tap water,38 andbottled water, where PFPrA was found to constitute up to 42% of totalPFAAs.39Regarding the inventory ofLake Vättern, most PFAAs weredetected in most of the samples. Similar concentrations were detectedat both locations, and there was no difference between samples collectedin spring compared to summer or between samples collected at 0.5 and30 m in the summer when the lake was stratified (4–26% relativestandard deviation). The measured concentrations are provided in 32 but muchlower in comparison to concentrations in Tangxun Lake, China , in 2011.40 Short-chain PFAAs accounted for the majority of PFAA contamination in samples from Tangxun Lake. Ultrashort-chainPFAAs were not included in that study.The concentrations of short-and long-chain PFAAs in surface watersamples from the lake are similar to concentrations across the GreatLakes between 2008 and 2017Figure S2. Only targetanalytes with a detection frequency of at least 50% in atmosphericdeposition or in surface water were included in the mass balance,and data below the LOD was treated as LOD/2. The input from the sewagetreatment plant and the paper mill that discharged directly into thelake was not reported separately but was combined and reported togetherwith the input from surface water inflow. This was done because theinput from the sampled sewage treatment plant and the paper mill wasnot representative for those sources to the lake, since some of thestreams receive effluent from industries and sewage treatment plants,and thus, input from effluents like these is already included in theinput from streams.The results of the massbalance of PFAAs in Lake Vätternare summarized in 3/year). Both atmospheric depositionand surface water inflow seem to be relevant sources for the inputof PFCAs to Lake Vättern, with atmospheric deposition accountingfor approximately 48% (PFOA) to 84% (PFDA) of the total input of thePFAAs included in the mass balance based on median estimates of inputvia surface water inflow. The largest input was observed for TFA andPFBA followed by PFPrA and PFOA, resulting in increases of 150, 4,1.6, and 1.5 kg/year, respectively.A net input of atleast 0.1 kg/year was calculated for all PFAAs except for PFHxS. Mostsurface water samples contained PFHxS in the same concentration range(0.56 ± 0.96 ng/L) as the inventory of Lake Vättern (0.69± 0.08 ng/L), and the zero net change is a result of the outflowbeing equal to the inflow ,PFOA (43–56%), PFDA (64–94%), and PFOS (39–56%)to Lake Vättern was somewhat higher than what has been observedin the Baltic Sea based on concentrations in atmospheric depositionsamples collected in 2007–2008 .2 (6400 km2 including the lake surface), atmosphericdeposition could be an important source of PFCAs to Lake Vätternvia surface water inflow. Based on the PFCA concentration in atmosphericdeposition and the amount of precipitation over the time of the study,the catchment area of Lake Vättern, excluding the lake surface,would receive an input of 230 kg/year (TFA), 3.5 kg/year (PFPrA),7.9 kg/year (PFBA), and 3.5 kg/year (PFOA). A certain amount is likelytaken up or retained by plants and soil,43 but an unknownfraction, which is larger for the smaller and more mobile PFCAs likeTFA, is most probably transported to the lake. This subject will befurther discussed in a later section.Considering the catchment area of 4500km7 A higher input was observed during the summermonths compared to the winter months, and a clear relationship betweenthe TFA input via atmospheric deposition and global radiation wasobserved . To assess the importance of atmosphericdegradation of precursor compounds to the lake, the relationship betweenatmospheric hydroxyl radical concentrations and precursor degradationproducts was investigated. Since hydroxyl radicals are produced by incomingglobal radiation into the atmosphere,44 the global radiation (W/m2), retrieved from measurementsby the Swedish Meteorological and Hydrological Institute, was usedto access the seasonal variations in hydroxyl radicals in the atmosphereover Lake Vättern. Significant positive monotonic correlationwas observed between the global radiation and the input of TFA , PFBA , PFHxA , and PFUnDA . Thisfinding suggests that hydroxyl radical-driven atmospheric oxidationof volatile precursors is an important source of PFCAs to the lakevia atmospheric deposition except for PFPeA and PFOA, for which othersources to the atmosphere might also play an important role.45The highest input viaatmospheric deposition was observed for TFA,which is a well-known degradation product of HFCs and HCFCs.observed 1.This fP < 0.01) except for PFOA. Seasonal trends of volatile PFASs anda relationship with temperature have previously been documented.46 In addition, seasonal differences in emissionrates of volatile precursors used in automobile and domestic air conditioningcan result in larger local and seasonal differences in TFA flux47 as a result of a shift from HFC-134a to HFO-1234yf,as the atmospheric lifetime with respect to oxidation by hydroxylradicals is considerably shorter for HFO-1234yf (∼11 days)48 compared to HFC-134a (∼14 years).49The flux of PFCAs was low relative to the global radiationduringMarch–May compared to June–August. This may be linkedto a lower atmospheric concentration of volatile precursors duringMarch–May, which can be influenced by factors such as temperatureand emission rates. Positive monotonic correlation was observed betweenthe flux of all PFCAs and temperature  to 90% (PFHpA) of the PFAAinput via surface water inflow based on median estimates . In general, higher input of both PFCAs and PFSAswas observed from streams with a higher flow rate with some exceptions2. In somTwo streams (streams1 and 13) were found to be important sourcesof PFPeA, PFHxS, and PFOS to Lake Vättern. Both streams eitherdirectly or indirectly receive stormwater from areas with known previoususe of AFFF. Stream 1 contributes with less than 0.1% of the waterinflow to the lake but account for 12, 16, and 13% of the input ofPFPeA, PFHxS, and PFOS, respectively, via surface water inflow . Stream 13 contributes with 21% of the water inflowand account for 68, 47, and 58% of the input of PFPeA, PFHxS, andPFOS, respectively, via surface water inflow . A third stream, which receives stormwater from a landfill,was identified as a source of PFPeA (stream 2), accounting for 2%of the input of PFPeA via surface water inflow .2, respectively) receiving a largeamount of atmospheric deposition resulting in an input of TFA of 88kg/year. A positive monotonic correlation was observed between TFAinput (kg/year) and catchment area (km3) . The other two main sources of TFA (streams12 and 17) only account for 4 and 2% of the surface water inflow,respectively, but account for 22 and 7% of the TFA input via surfacewater inflow . Stream 12 has a relativelylarge catchment area (420 km2), while the catchment areaof stream 17 is relatively low (84 km2). These two streamsare not subject to any known contamination sources but are surroundedby agricultural activity. TFA has been identified as a main metaboliteof pesticides containing a trifluoromethyl structure.51 Agriculture could therefore potentially be an important source ofTFA contamination, but this could not be verified in the present project,and from the present data, atmospheric deposition seems to be thedominant route for TFA contamination.Five streams were identified as major sources of TFA 2. Three 3) and the input (kg/year) of allPFCAs  except PFPeA,PFHxA, and PFHpA. Among the PFSAs, only TFMS input was correlatedwith the catchment area .The correlation between the input and the catchment area might alsobe associated with a larger number of local sources such as sewagetreatment plants.Both surface water inflowand atmospheric deposition were foundto be important sources of PFAAs to Lake Vättern. Atmosphericdeposition seemed to be the main pathway for input of most PFCAs,including TFA. The input of TFA is also hypothesized to originatefrom agriculture, although the role of fluorine-containing pesticidesas a source of contamination of freshwater lakes needs further investigation.From the catchment area of Lake Vättern, it is also possiblethat atmospheric deposition could account for PFAA input via surfacewater inflow. Positive monotonic correlation was observed betweenthe catchment area , while PFOS, PFOA, PFNA and PFDA concentrationsdecreased over time, which is suggested as a result of phaseouts andregulatory measures.32 PFBA has previouslybeen suggested to be uniformly distributed in the atmosphere.52 Here, we suggest future monitoring of TFA andPFPrA to confirm that ultrashort-chain PFAAs are also distributedin the global atmosphere. Local anthropogenic sources seem to havea higher influence on most PFSAs, including ultrashort-chain PFSAs.The observed correlations between global radiationand the PFCAinput indicate that atmospheric oxidation of volatile precursors isa major source of C5 Other transformation mechanisms in water arenot known. Sedimentation as well as uptake in biota could be an importantsink for long-chain PFAAs5 but are notconsidered relevant for ultrashort-chain PFAAs due to the high polarityof these compounds. The relevance of volatilization or potential transferto the atmosphere via evaporation or formation of aerosols from thebreaking of waves is not known.The main output was assumed to be via the main outflow. Other outputpathways such as transformation, chemical and/or photochemical degradationin the water column, sedimentation, and volatilization were consideredof minor importance and not included in the study. Phototransformationin the water column was found to only account for 0.03–0.4%of the total output of PFHxA, PFOA, PFDA, and PFOS in the Baltic Sea.53 especially for these highly polar compounds requiring combinationsof advanced water treatment systems for their removal.54The estimated input of severalof the measured PFAAs to Lake Vätternexceeds the output. This positive net change will result in increasedPFAA concentration in the water column over time. The doubling timefor the PFAA concentration in the lake can be estimated by comparingthe net change with the inventory in the lake, which is relevant forTFA (15–22 years), PFPRA (19–29 years), and PFBA (9–17years) based on low- and high-bound estimates. Accumulation of persistentanthropogenic compounds in drinking water sources is a cause of concernsolely based on the persistence criteria,32 Alarger set of samples would, in addition to less uncertainty in thereported concentration ranges, allow for detection of seasonal variationsin PFAA input via inflowing streams that could possibly be attributedto agricultural activities and/or seasonal variations in vegetation.Since ultrashort-chain PFAAs can be taken up by plants,19 the input via inflowing streams could follow the life cycle of vegetation,with larger input being associated with decomposition during fall.The current study has assessed the relevanceof atmospheric depositionand surface water inflow to PFAA contamination in a large drinkingwater source in Sweden over a 1 year time period, including inputfrom 19 streams and atmospheric deposition at one site, with the assumptionthat the composition and amount of deposition is equal over the entirelake. Precipitation samples across the Great Lakes show that short-chainPFAA are evenly distributed in the global atmosphere, while concentrationsof PFOS and PFOA are greater in urbanized/industrialized areas ascompared to more remote locations.The positive net input of PFAAs to Lake Vättern warrantsfurther monitoring of these compounds. Precipitation and surface wateris shown here as suitable matrices for monitoring of short- and ultrashort-chainPFAA, providing additional data currently scarce in the scientificliterature.


Necrotizing enterocolitis (NEC) is one of the most important causes of morbidity and mortality in premature infants. Although there are no specific diagnostic tools, the main factors affecting prognosis are clinical and laboratory findings, and early diagnosis and treatment. In this study, we demonstrate the importance of dual-energy computed tomography (DECT) in confirming intestinal ischemia in neonates with NEC.Patients diagnosed with NEC in a neonatal intensive care unit were staged according to modified Bell’s classification, and DECT was performed on patients with NEC stages 2-A, 2-B and 3-A. According to their DECT reports, these patients were then separated into two groups: those with intestinal ischemia and those without intestinal ischemia. The patients with intestinal ischemia were evaluated using surgical reports, and the other patients were evaluated using clinical findings.DECT was performed in 21 patients with NEC stages 2-A, 2-B and 3-A. Twelve patients (57.1%) without ischemia were followed up without surgery. Nine patients (42.9%) with ischemia on DECT were operated on, and resection and anastomosis or ileostomy and colostomy were performed. In patients with NEC, DECT significantly increases overall diagnostic confidence in assessing intestinal necrosis when compared with traditional diagnostic methods. NEC is one of the most important causes of morbidity and mortality in neonatal intensive care units [1–3]. It affects 4%–13% of infants have low birth weight (<1500 g) and 1%–8% of infants admitted to neonatal intensive care units . Clinical findings are manifested in a variety of ways, including feed intolerance, prefeed residue with bile-stained aspirates and/or vomit, bradycardia, fulminant shock, and death. Plain antero-posterior and left lateral decubitis radiographs are the cornerstone of NEC diagnosis. According to clinical severity, pneumatosis intestinalis (PI), portal vein gas (PVG), pneuomoperitoneum (PP), intraperitoneal fluid, and persistently dilated fixed loops are some of the associated findings can be seen on plain abdominal graphy (PAG) [6]. Ideally, surgery should not be performed until gangrene is present; however, it should be undertaken before perforation occurs.Dual-energy computed tomography (DECT) is a new technique that has the capability of differentiating matter and tissue according to CT density values obtained from two synchronous CT acquisitions at different tube potentials. It should be emphasized that described systems can also be used for simply clinical routine examination requiring standard single-energy mode DECT acquisition [7]. Dual-source CT system is used for the evaluation of renal masses, liver lesions, urinary calculi, small bowel, pancreas, and adrenal glands [8–11]. This study is the first report on the use of DECT as a diagnostic modality in patients with NEC. DECT will enable better detection of hypovascular segments of the bowel that enhance poorly. DECT makes it possible to distinguish different types of matter that cause high-density areas in and around the bowel. In this study, we aimed to detect intestinal ischemia in newborns with NEC using a novel method. Therefore, we think that surgical decision-making processes of these patients before bowel perforation may be easier and may provide positive contributions to patients in terms of morbidity and mortality.Over a period of 36 months, 34 consecutive patients with a clinical diagnosis of NEC were enrolled in this observational study. The study was approved by the medical ethics committee of our hospital (03.12.2015/10). The clinical and laboratory findings were recorded. The initial diagnosis of NEC was clinical and based mainly on signs and symptoms such as abdominal distension, gastric residue, vomiting (feeding intolerance), lack of stool, diarrhea, hematochezia, abdominal wall erythema, and palpable abdominal mass. All patients were assessed using plain abdominal radiography, and the radiographs were explicated by pediatric radiologists. The radiological criteria evaluated as predictors of NEC on plain abdominal radiographs were PI, PVG, PP, fixed intestinal loops, gasless abdomen, and peritoneal fluid. Modified Bell’s classification was used to determine the stage of the disease [4]. Each patient was scanned with DECT, except for those whose clinical condition made it difficult to transport them to the radiology unit and those classified as Bell’s stage 1-A, 1-B, 3-B, since it is unacceptable to expose newborns to radiation unnecessarily. DECT results were recorded as showing necrosis to be present or not present. Indications for surgical treatment were clinical or radiological evidence of intestinal perforation, abdominal wall erythema/edema indicating gangrene, presence of abdominal palpable mass, failure to respond to conservative management with a rapid decline in health status, and necrosis on DECT .The DECT examinations were carried out by using a 256-slice dual-source CT . Iopromide  followed by 30 mL of saline was injected into the antecubital vein at a flow rate of 3 mL/s. A bolus tracking technique was used, and the region of interest was located in the abdominal aorta. The following scanning protocols were carried out: tube voltage of 100 kVp/Sn 140 kV (140 kV with tin filter); automatic mAs current (by CARE Dose4D); 64 × 0.6 mm collimation; rotation time of 0.28 seconds; automatic pitch; cranio-caudal scan direction; slice width 0.6 mm; increment 0.3 mm; kernel D33f abdominal view; and temporal resolution of 75/140/330 ms. The CT dose index volume and dose length product of the DECT scans were recorded for each patient protocol. High or low voltage data were reconstructed separately to analyze of abdominal perfusion. To optimize signal/noise ratio, dual-energy convolution kernel (D30f) is used, which is composed of 140 ms temporal resolution with 1.5 mm thickness and 1 mm increments. A specific platform  was used for extended analysis of the reconstructed data sets. Tissue data analyzed with 0.6 mm same sections reconstructed with 330 ms temporal resolution, dedicated algorithm of each abdominal perfusion blood volume for the evaluation of abdominal perfusion incorporation obtaining dual-energy image of post processing software application . Two radiologists  were blinded to the clinical data. They examined the CT images, and iodine map images were obtained from 21 examinations independently; in case of disagreement, a final decision was reached by consensus. The dark areas on the iodine map were considered to be necrotic .The data were analyzed using SPSS 15.0 software, and the Kolmogorov–Smirnov test was applied. Percentages, mean values, and standard deviations were used as identifier statistical values. Specificity, sensitivity, positive/negative predictive values, and diagnostic accuracy rates were calculated for clinical and radiological criteria, and statistical significance was observed for diagnostic tests using standard epidemiological measures. Patients who received surgery were evaluated in terms of surgical outcomes, while nonsurgical patients were evaluated in terms of clinical results.Data were analyzed from 34 patients  with a diagnosis of NEC who were followed between October 2015 and October 2018. The mean birth weeks of these patients were 30.12+/−3.98 weeks, and their mean birth weights were 1306.21+/−527.46 grams. Five patients had stage 1 NEC , 22 patients had stage 2 NEC , and seven patients had stage 3 NEC  according to modified Bell’s classification (Table 1). The clinical and laboratory findings of the patients were recorded. The clinical and laboratory findings are shown in Table 1. DECT was not performed to patients with stage 3-B and all stage 1 NEC patients. DECT was performed in 21 patients with stages 2-A (9 patients), 2-B (9 patients) and 3-A (3 patients) . Ischemia was detected in nine patients  using DECT  and confirmed at surgery in all of these patients. Seven patients had PI, four had PVG, six had fixed intestinal loop on PAG. Three patients had abdominal skin erythema . Ostomy or end-to-end anastomosis was performed after resection for ischemic segments at operation. Histologic assessment of the resected specimens confirmed bowel ischemia in all . Our study found DECT to have 100% sensitivity and 100% specificity for detecting bowel ischemia and 100% positive predictive value (Table 2). Bowel ischemia was not detected in 12 patients  on DECT . Five of these patients had PI, three had PVG, and two had fixed intestinal loop on PAG . A patient who had PVG on PAG was later verified on DECT as having portal venous thrombus . Nine of these 12 patients were discharged uneventfully. One patient who received treatment for portal venous thrombus died of pulmonary and intracranial hemorrhage, another died of cardiac pathologies , and another died of pulmonary hemorrhage related to respiratory distress syndrome.We calculated the following statistical values for PI: sensitivity 61.5%, specificity 57.1%, positive predictive value 47.1%, negative predictive value 70.6%, and diagnostic accuracy 0.62. We calculated the following statistical values for PVG: sensitivity 38.5%, specificity 76.2%, positive predictive value 50.0%, negative predictive value 66.7%, and diagnostic accuracy 0.65 (Table 2).The effective radiation dose of DECT was calculated using a method proposed by the American Association of Physics in Medicine for the measurement, reporting, and management of radiation doses in CT. Using the dose length product and a conversion coefficient for the neonate abdomen (k = 0.049 mSv/[mGy cm]), the mean effective radiation dose for DECT was calculated as 2.21 ± 0.24 mSv.NEC is one of the most common and most serious acquired bowel diseases in premature newborns. Over the past 20 years, great improvements in neonatal medicine have resulted in improved results. In spite of the decrease of NEC morbidity and mortality, the incidence of NEC is increasing [12]. Early diagnosis and treatment are the main factors affecting prognosis; in particular, diagnosis of ischemia in clinically suspected patients is extremely important in improving mortality rates . Today’s practice is limited to a few nonspecific markers of systemic disease . Although most of the physicians have tried to understand the etiology of NEC for early diagnosis and effective treatment; immunological markers of the disease should also be examined [14]. Evenett et al. conducted a systematic review of previous studies of serological tests to establish which of them are useful in the diagnosis of NEC [15]. Their findings suggested that intestinal fatty-acid binding protein and platelet‑activating factor have sensitivity and specificity in the diagnosis of NEC. Nevertheless, for more reliable results, these indicators should be tested in further prospective studies with larger sample groups [15].Most surgeons rely on radiological signs to establish a diagnosis of NEC. Plain radiographs are essential for the radiological diagnosis of NEC, but the specificity and sensitivity of this method are relatively weak. PI (cystic or linear) is the most specific radiographic finding. Other common radiographic findings are gas-filled loops of bowel, persistently dilated loops of bowel, air-fluid levels, thickened bowel walls, PVG, and PP. PI is a pathognomonic sign of NEC in a true clinical setting. However, the level of PI does not correspond in every case with the severity of the disease [16–18], and Alda and colleagues reported the sensitivity of PI findings as lower than 50% [6]. In our study, DECT was performed in 12 of 16 patients with PI. Intestinal ischemia was not determined in five of these 12 patients, and four of the five were discharged without any problems. A 25-week-old patient died of cardiac and respiratory failure.PVG, another hallmark of NEC, is a less common radiographic sign associated with higher mortality rates and severe disease . Nonetheless, with medical management, many patients with PVG recover fully [20]. Karl Muchantef and colleagues commented on sonographic and radiologic images for 55 patients, and they did not accept PVG and intramural gas as absolute surgical indications [21]. The results of the present study are in line with these observations, showing that air in the portal system was the variable associated with a high risk of intestinal necrosis. DECT was performed in seven of the 10 patients, and intestinal necrosis was found in only four of them. PVG had a positive predictive value of 50%, a negative predictive value of 66.7%, and a diagnostic accuracy rate of 0.65.The presence of PP on radiograph is accepted as an absolute indication for surgery . However, PP has been reported in approximately 50% to 75% of patients with perforation . In cases where clinicians wait for evidence of PP before deciding to perform surgery, it may be far too late for the patient, especially for an extremely premature infant.Laparoscopy was used as a method to identify necrotic bowel segments and to decide requiring in NEC . It has been experimentally shown that fluorescein laparoscopy is superior to another diagnostic methods to identify early stage of NEC and to distinguish reversible and irreversible intestinal ischemia [26–28]. Recently, Numanoğlu and colleagues have emphasized that this method increases diagnostic accuracy rate of intestinal ischemia in suspected NEC patients [29]. However, this study was performed on only 13 patients, and laparotomy was performed in 10 of these patients due to detected intestinal ischemia. Negative laparoscopy does not seem to be acceptable in patients undergoing anesthesia, such as suspected NEC, in patients whose general condition is extremely impaired and other system anomalies are frequently observed.On conventional CT, it is not generally possible to appreciate the signs of reduced or absent mural enhancement in cases of intramural hemorrhage or early ischemia, or when mural enhancement is suboptimal because of high image noise or low IV contrast concentration. It has been suggested that CT, contrast examinations of the gastrointestinal tract, and magnetic resonance imaging are not clinically useful modalities for the treatment of infants with NEC [20]. The most important reasons for this are the high radiation rates of CT and the frequent possibility of diagnosing NEC by means of physical examination and other modalities. According to the literature, the mean radiation dose for CT for pediatric patients is between 5.7 and 6.2 mSv [30]. Our radiation dose (2.21 ± 0.24 mSv) is lower than literature and equivalent to about 20 plain radiographs [30]. Although our doses are within normal limits.It has been shown that DECT mostly specific to detect of hypovascular solid organ lesions such as liver and pancreas. In this respect, it is thought that this technique will enable better detection of hypovascular segments of the bowel that enhance poorly. It also has the ability to emphasize mural hyper-enhancement or hypo-enhancement. Primary preclinical studies have indicated that DECT has significant potential to describe intestinal infarction [31]. Moreover, when ischemia in small intestinal obstruction is evaluated using low‑keV virtual monoenergetic images, early clinical results have suggested an increase in diagnostic confidence [32]. Subsequently, Lourenco has reported that iodine map and 40 keV monoenergetic images increase the conspicuity of acute intestinal ischemia, resulting in improved diagnostic accuracy [33]. The use of low-keV virtual mono-energetic images mostly detect increased vascularization of the inflamed intestinal wall such as Crohn’s disease [32]. As active peristalsis of the bowel mistakenly considered as wall hyperemia on CT imaging, iodine density images should be used to eliminate of artefacts [34]. However, as yet there has been no study in English of DECT and its beneficial effects in cases of NEC.In this study, we found that all patients with intestinal ischemia on DECT also had intestinal ischemia at surgery. The patients without ischemia on DECT examination were treated medically, and nine of them were discharged without any problems. Three patients died of nongastrointestinal causes. We found that DECT had a diagnostic accuracy rate of 100% for bowel ischemia. There are some limitations in this study. It should be borne in mind that infants with advanced NEC almost always need mechanical ventilator support and high levels of inotropic support. For that reason, in a hospital where the radiological unit is situated some distance from the intensive care unit, transportation of such patients for DECT observation may be highly problematic, and serious difficulties may be experienced. Even if the radiological unit is close to the intensive care unit, significant clinical deterioration may occur during the transportation of an intubated infant under inotropic support. Moreover, it is very well known that some patients may have transient intestinal ischemia that resolves with bowel rest and antibiotic therapy . On the other hand, we also know that mild ischemia may only involve the mucosa and that it takes more to cause full-thickness necrosis. In this clinical study, we have no evidence that the study can quantitate the degree of ischemia. So, we need to design a study in rats by using a NEC model in which different degrees of ischemia could be induced. Only, with such a study, we can know whether DECT can demonstrate the presence of a mild ischemia and whether these infants with DECT positive for mucosal ischemia would have got away without surgery. In NEC patients, the use of DECT may help to improve early detection of bowel ischemia by increasing the conspicuity of under perfused tissues on low-kiloelectron-volt and iodine material density images. Additional investigation of DECT must be carried out in larger series to evaluate its potential to improve the sensitivity and specificity of conventional radiological methods for detecting early small bowel ischemia.The authors declare that no funding was received.The study was approved by the medical ethics committee of our hospital (03.12.2015/10). All procedures involving human beings were conducted following the ethical standards and principles outlined in the Helsinki Declaration (2008).


Pseudomonas sp. monoculture here. This work challenges the binary classification of FOM as either allochthonous or autochthonous, suggesting that FOM processing and production occurs along a dynamic continuum. Within this study, fluorescence intensity data for the environmental bacteria isolate monocultures are presented as enumeration corrected data, for the first time providing quantitative fluorescence data per bacterial colony forming unit (cfu). From this, we are able to assess the relative contribution of different bacteria to the autochthonous FOM pool and if this material is cellular or extracellular.This work investigates the origin and range of fluorescent organic matter (FOM) produced in-situ by environmentally sourced freshwater bacteria. Aquatic FOM is an essential component in global carbon cycling and is generally classified as either autochthonous, produced in-situ via microbial processes, or allochthonous, transported into aquatic systems from external sources. We have demonstrated that, within laboratory model systems, environmentally sourced mixed microbial communities and bacterial isolates can produce and/or export FOM associated with both autochthonous and allochthonous material. This study focuses on fluorescence peak B, T, M, C and C+, exploring (1) the cellular nature of FOM produced, (2) FOM exported as extracellular material into the water column and (3) the impact of physical cell lysis on FOM signature. For the laboratory model systems studied, Peak T fluorescence is retained within bacterial cells (>68%), while Peak C fluorescence is mainly observed as extracellular material (>80%). Peak M is identified as both cellular and extracellular FOM, produced by all isolated freshwater microorganisms investigated. The origin of Peak C+ is postulated to originate from functional metabolites associated with specific microorganisms, seen specifically within the  DOM is a highly complex chemical composite , and quasolution ,4. This solution ,6,7,8.Fluorescent organic matter (FOM), the naturally fluorescent fraction of DOM, has been characterised across a range of aquatic systems ,9,10,11.Classifying FOM in this way has enabled the study of the variable temporal and spatial detection of these fluorescent signatures within aquatic systems, as indicators of water quality . FreshwaAlthough there has been some exploration of the autochthonous origin of microbial FOM outside of marine research, this has been limited to the exploration of single bacterial species  or has uThe aim of this study was to investigate the in-situ production of FOM by isolated freshwater microorganisms. Furthermore, this work challenges current assumptions that freshwater FOM is perceived to be either autochthonous or allochthonous in origin. An improved understanding of the role of environmental microorganisms in the production of FOM in freshwaters will help address current knowledge gaps regarding the importance of surface freshwater systems within global carbon cycling.An environmental freshwater sample was enriched and grown to assess the role of mixed microbial communities in the production of FOM. Samples were analysed using fluorescent spectroscopy to produce Excitation Emission Matrices (EEM) spectra, which enabled investigation of the resultant FOM. The use of the microbial community within a model system provides a snapshot of the potential community and FOM production from this system. Bacteria from this environmental freshwater sample were also isolated, cultured and subjected to the same analysis as the mixed freshwater microbial community. The environmentally derived bacterial monocultures allowed for a more in-depth exploration of the FOM production at an individual environmental isolate level. All culture samples were fractioned prior to analysis to elucidate the microbial origin of FOM production at a cellular level in freshwater systems, i.e., whether FOM is associated with cellular material or is produced and then exported by microorganisms into the water system.A water sample was collected from an environmental freshwater body located at the University of the West of England, Bristol (UWE), UK . Water was collected at a depth of ~30 cm from the surface, and ~3 m from the edge of the waterbody. Samples were collected using a five litre HDPE container, cleaned with a 1% Virkon ™  solution, and then rinsed thoroughly with deionised water to prevent chemical or biological contamination. The container was rinsed three times with sample water immediately prior to collection. Details of the physicochemical data for the freshwater body on the day of collection can be found in the g and washed three times in osmotically stable ¼ strength Ringer’s solution , to remove media/supernatant and organic matter. The cell pellets were then resuspended in 10 mL of the minimal media and used as the microbial inoculum for FOM production experiments.The environmental freshwater sample  was filt® 0.2 µm nitrocellulose membrane filters, GE Healthcare, Chalfont St Giles, UK). These filters were placed on selective and differential agar plates and incubated overnight (24 h) at 30 °C to culture a range of environmental bacteria. Twelve single colonies were identified from the plates, and sub-cultured on nutrient agar plates for 24 h at 30 °C: five colonies were taken from the R2A agar  plates; four colonies taken from the Brilliance ™ E. coli/Coliform  plates, which were presumptive E. coli and presumptive coliforms; and three colonies selected from the Difco ™ Pseudomonas Isolation Agar  plates, two of which were presumptive Pseudomonas sp.Aliquots (100 mL) of the environmental freshwater sample were vacuum filtered through sterile 0.2 µm filters  . Prior tBacillus sp. ; Enterobacter sp. ; Escherichia coli ; Pseudomonas sp. ; and Staphylococcus sp. .The five environmental bacterial isolates that were successfully identified were: v/v glucose solution, with sources of phosphate, nitrogen, sodium and magnesium [2  and trace elements (concentration 0.1% v/v) [A non-fluorescent minimal medium, containing no proteinaceous material, was used to promote growth of the cultures within our laboratory model system. The basal medium contained a 0.2% agnesium , CaCl2 (.1% v/v) . This waOvernight 20 mL cultures (24 h), for both the environmental microbial community inoculum and the environmental bacterial isolate monocultures, were incubated at 30 °C with shaking (150 rpm). The overnight cultures were then fractionated to provide information about cellular and extracellular FOM and to investigate the contribution of cell lysis to the FOM signal . The ove−1). Enumeration for each bacterial isolate was repeated three times, with each biological replicate also being enumerated in triplicate.The overnight monocultures for the five environmental bacterial isolates, detailed in ® . Bacterial supernatant samples were filtered using sterile Minisart® 0.2 μm cellulose syringe filters  to guarantee that all cells were removed before being analysed. Cellular samples (resuspended and lysed cells) were not filtered prior to spectroscopic analysis to ensure sample integrity [ex 240–500 nm at 1 nm steps, λem 247.88–829.85 nm in 1.16 nm steps, with an integration time of 500 ms. A micro quartz cuvette (1400 μL) with a 10 mm path-length was used throughout.Fluorescence excitation emission matrices (EEMs) of the overnight culture subsamples were collected using an Aqualogntegrity ,50. The ex = λem and 2λex = λem) [® software . All fluorescence intensity data were converted and reported in quinine sulphate units (QSU) (1 QSU = 1 µg−1 quinine sulphate) [TM .All fluorescence spectra were blank subtracted using sterile media, corrected for inner filter effects (for both excitation and emission wavelengths) and first and second order Rayleigh Scattering masked (±10 nm at λx = λem) ,39,40,51ulphate) ,53 via aTM , was used to create the EEM maps and to undertake the peak picking for specific fluorescence regions of interest [ex 240–490 nm, λem 250–550 nm. EEM data were also investigated by employing parallel factor (PARAFAC) analysis [® PLS-Toolbox .A custom script, written in Pythoninterest ,40. The analysis  in Solo 10 cfu mL−1. To simplify the data and enhance visual interpretation, the data have been converted to log numbers as is standard practice for microbial counts. Consequently, monoculture fluorescence data are expressed as “Log Normalised QSU”. For the environmental microbial community culture, the fluorescence intensity data for all sample fractions are not normalised for cell density, due to the limitations of representatively culturing complete environmental bacterial communities.Fluorescence intensity data for all sample fractions of the environmental bacterial isolate monocultures have been cell density normalised, obtained from viable counts , wherebyp value of <0.05 was regarded as significant. All statistics were performed in Prism 9 .The statistical significance of variation between fluorescence peak intensity data, obtained via peak picking, for the different sample fractions was undertaken using a two-way ANOVA. A paired t-test was conducted to determine if there was a significant difference between the extracellular (supernatant fraction) and the cellular (both resuspended cell and lysed cell fractions) locations of the fluorescence peak intensity data. A This work investigated the in-situ production of FOM by mixed environmental microbial communities and environmental bacterial isolates using a laboratory model system. The environmental microbial community culture and environmental bacterial isolates used here were obtained from the same water sample. The use of the microbial community within this model system provides a “snapshot” of the FOM production within this system, whilst the bacterial isolates allow for a more in-depth exploration of the FOM production at an individual environmental isolate level within the laboratory model system.n = 234) nor the bacterial isolates data (n = 268). This is likely to be due to the dominance and ubiquitous nature of certain fluorescence peaks, namely peaks T and C. Subsequently, all EEM data obtained from the overnight cultures were subjected to peak picking, an established method for spectral analysis [PARAFAC analysis was performed on the EEM datasets obtained from the freshwater model system. This analysis was unable to provide a robust model, CORCONDIA > 90% , for theanalysis . The fluAll peaks detailed in n = 3, ±SD). Samples were interrogated to explore the cellular and extracellular nature of the microbially derived FOM. logQSU) for the fluorescence peaks investigated for all sample fractions, cellular (resuspended and the lysed cell fractions) and extracellular (supernatant), of the microbial community culture.The freshwater environmental microbial community was cultured overnight (24 h) to gain insights into potential FOM production by the total microbial community within the laboratory freshwater system. Significant increases in the total fluorescence intensity for all peaks  were seen from the point of inoculation to the 24 h timepoint. The percentage increase in the complete sample fluorescence intensities (without fractionation) during the 24 h incubation for each peak was: Peak B, 1573 ± 54%; Peak T, 1606 ± 90%; Peak M, 4036 ± 1343%; Peak C, 45,315 ± 9095%; Peak C+, 135,707 ± 11,868%  to obtain a detailed understanding of the potential for different environmental bacterial isolates to contribute to FOM production within environmental freshwater systems. The excitation emission matrices for the complete sample (prior to fractionation) of each of the five bacterial isolates are shown in Bacillus sp., E. coli and Staphylococcus sp. monocultures . In contrast, Peak B fluorescence increased for the Enterobacter sp. and Pseudomonas sp. monocultures . The largest increase in fluorescence intensities was seen at wavelengths >300 nm, indicating that the observed increases in Peak B for Enterobacter sp. and Pseudomonas sp. are associated with the presence of high Peak T fluorescence intensities.The presence of Peak B within the different investigated monocultures varied considerably. Peak B fluorescence was associated with cellular material within all five environmental bacterial isolates. Peak B fluorescence observed in the complete sample decreased from initial inoculation to 24 h for the E. coli and Staphylococcus sp. cells exhibit higher total relative fluorescence than Pseudomonas sp., Enterobacter sp. and Bacillus sp.E. coli, Staphylococcus sp., Enterobacter sp. and Bacillus sp. (Pseudomonas sp. due to the high intensity of Peak C+. Peak T is observed as predominantly cellular (resuspended and lysed cells), accounting for between 68 and 92% of Peak T fluorescence intensity for the five bacterial isolates investigated (p < 0.0001) between the Peak T intensity when analysing all fractions  for all five overnight monocultures. Further analysis was undertaken by classifying the resuspended and lysed cell sample fractions as cellular, and the supernatant as extracellular material. Based on this classification, a paired-samples t-test was undertaken to explore the difference between cellular and extracellular FOM for each isolated species. This revealed a significant difference (p < 0.05) between cellular (resuspended and lysed cells) and extracellular (supernatant) Peak T across all species  in the Peak M intensity, with a paired t-test revealing significant differences (p < 0.05) between extracellular (supernatant) and cellular (resuspended and lysed cells) Peak M for the Enterobacter sp. and E. coli isolates only.Peak M, shown in p < 0.001) between the Peak C intensity when analysing all fractions  for all five overnight monocultures. A paired-samples t-test revealed a significant difference (p < 0.05) between cellular (resuspended and lysed cells) and extracellular (supernatant) Peak C for Bacillus sp., Enterobacter sp., E. coli and Pseudomonas sp. Due to variability in the data, no significant difference (p > 0.05) between extracellular and cellular Peak C was identified for Staphylococcus sp.; Peak C contributes <3.5% of the relative fluorescence for this bacterial isolate . The other species also exhibit Peak C+ fluorescence in both cellular and extracellular material albeit significantly lower, <3%, in terms of its overall contribution to the total fluorescence intensity (p < 0.0001) between the Peak C+ intensity for all fractions  from all five bacterial isolate monocultures. A paired t-test revealed a significant difference (p < 0.01) between extracellular (supernatant) and cellular (resuspended and lysed cells) Peak C+ for Enterobacter sp., E. coli and Pseudomonas sp. No significant difference (p > 0.05) between extracellular and cellular Peak C+ was identified for Bacillus sp. and Staphylococcus sp., which only contributes <3% of the relative fluorescence for these bacterial isolates  and also exported from cells (extracellular).Peaks B and T have been frequently characterised within a range of aquatic environments ,38,57,58The presence of Peak T is identified in all sample fractions associated with the microbial community cultures . There iPeak M is discussed widely in the literature as autochthonous FOM associated with marine biodegraded organic matter ,57,64,65Peaks C and C+ are associated with high molecular weight, terrestrially derived, allochthonous organic material ,20,26. PIn our system, the majority of Peak C+ FOM produced by the microbial community is extracellular. Previous literature has suggested that Peak C+ fluorescence may be associated with exotoxin production by specific microorganisms ,67. We oFOM peaks, such as peaks T, C and M, are common across a range of different freshwater systems. To explore the universal presence of this FOM further and to better understand FOM production from a freshwater microbial community, individual environmental bacterial isolates were investigated. The isolated species were cultured from the same freshwater source (sample) as the microbial community and grown, as monocultures, in the same laboratory model and conditions.Staphylococcus sp. and E. coli were shown to produce the most fluorescence per cfu (colony forming unit), see The data presented here demonstrate that all of the environmental bacterial isolates studied are capable of producing a range of FOM, in relation to both the location (peak) and intensity. Within this laboratory system, It has been widely postulated that cell lysis and biodegradation are the mechanisms by which higher molecular weight autochthonous material is derived within marine systems ,38,64,68Peak T fluorescence is identified within the FOM produced by all environmental bacterial isolates studied. In all cases, the highest intensities of Peak T were observed in the resuspended and lysed cells a, with >Bacillus sp., E. coli and Staphylococcus sp. monocultures. This could be explained by the highly labile nature of Peak B FOM, meaning it may be rapidly utilised (<24 h) within the closed monoculture laboratory system investigated here. Furthermore, this FOM has been demonstrated to be more vulnerable to quenching effects, such as energy transfer, depolarisation, oxygen quenching and protein conformation [Peak B, similarly to Peak T, is associated with amino acids, specifically tyrosine ,32,57,75ormation ,78,79,80Peak M was ubiquitous within all sample fractions (cellular and extracellular) for all of the environmental bacterial isolates b. AlthouPeak C-related FOM was ubiquitous across all sample fractions for all environmental bacterial isolate monocultures c, althouPseudomonas sp.  with the siderophore pyoverdine, a high molecular weight (1365 Da) extracellular iron-scavenging metabolite [High fluorescence intensity of Peak C+ was identified within the supernatant sample fraction for onas sp. d, with eonas sp. . The domtabolite ,67. FurtThe FOM data from the bacterial isolates clearly demonstrate that freshwater bacteria can produce a range of FOM, including fluorescent compounds associated with higher molecular weight ,36,84,85The use of a laboratory model presents the opportunity to interrogate culturable environmentally sourced bacterial isolates and communities in controlled conditions. This approach offers insights into FOM production by these microorganisms, albeit with limitations. These limitations include the role and impact of uncultured bacteria on the FOM signature and composition. To further the understanding of microbial–FOM interactions, a more representative laboratory model could be employed with the aim of limiting preferential growth conditions, for example by using a simulated freshwater matrix instead of a growth media. Alongside this, the continued use of different environmentally derived freshwater microorganisms, including the exploration of algae, fungi, viruses and other bacteria, would greatly enhance the current knowledge base surrounding microbial interactions with freshwater FOM and how this impacts global biogeochemical cycling.In addition to the development of more representative model systems, further understanding of FOM production and transformation could be gained through the use of high-resolution analytical techniques, such as FTICR-MS, Orbitrap-MS or metabolomics, in tandem with fluorescence spectroscopy. The use of such techniques, specifically FTICR-MS, is used for the quantification of FOM components that have been identified and derived from parallel factor (PARAFAC) analysis . HoweverThis work has, at the very least, questioned the binary classification of allochthonous or autochthonous FOM as currently applied to freshwater systems. Environmental freshwater microbes produce a range of FOM in-situ , both processing and producing FOM along a dynamic continuum, albeit demonstrated in a laboratory freshwater model system. The microbial production and potential export of metabolic by-products and/or the production of functional proteins are possible reasons for the observed FOM characteristics. Understanding the mechanisms for this will provide knowledge and insight regarding microbial processing and its relevance to carbon cycling on a global scale.This work explicitly explores the potential contribution of microbial FOM production and the impact of cell lysis on the composition and intensity of freshwater FOM. The lysed cell fractions exhibit similar fluorescence characteristics to intact cells, but do demonstrate elevated fluorescence intensity in the lysed cells fraction for both peaks C and M. This demonstrates that cell lysis can contribute to FOM and the carbon pool, but is unlikely to be the sole source of ‘microbial’ Peak C and Peak M. However, further work regarding the role cell lysis plays in freshwater FOM transformation and carbon cycling in dynamic active environmental microbial communities, and over time, is needed.This study identifies the production of Peak B by the environmental microbial community, but this is not reflected by the individual bacterial isolates. Interactions within the microbial community, or community members (not isolated here), could be responsible for the Peak B FOM signatures seen. This may also be explained by the use of bacterial monocultures here, something that does not exist in nature, which may have both produced and processed Peak B FOM, thereby rapidly preventing the identification of this labile FOM after the 24 h culturing period. The frequent, but not universal, presence of Peak B in environmental freshwater systems highlights further exploration of this phenomenon as essential for understanding labile FOM interaction and the potential impacts this may have on the global carbon cycle.This work confirms the microbial origin of Peak T. The presence of Peak T fluorescence is ubiquitous in all sample fractions, being identified at significantly higher fluorescence intensities within cellular sample fractions. Further work is required to understand the mechanisms by which this FOM is produced and exported into freshwater systems. Peak C and M fluorescence are universally produced within all sample fractions, but Peak C is predominantly found in the supernatant sample fraction. This suggests that the majority of Peak C is exported as extracellular material into the surrounding system. These results support the findings of previous work that investigated bacterial reference strains. Importantly, this work demonstrates that environmental freshwater microbes also produce and export a range of FOM. This knowledge could have implications for enhancing our understanding of the role that freshwater systems play in the global cycling of carbon.


In both groups the research for thyroid antibodies, anti-thyroid peroxidase antibodies and anti-thyroglobulin antibodies, was performed. The positivity had a prevalence of 17.54% in women with autoimmune diseases (n = 47) versus 5.57% in the control group (n = 64) . Only major rheumatic diseases (MRD) were analysed for pregnancy outcome : systemic lupus erythematosus (SLE) n = 36, antiphospholipid syndrome (APS) n = 44 and connective tissue diseases (CTD) n = 23. MRD were divided according to positive or negative results for thyroid antibodies. Thyroiditis in CDT patients showed a detrimental effect on pregnancy outcome, in terms of earlier week of delivery: 37.86 ± 0.90 (mean ± SD) in CTD with thyroiditis versus 38.56 ± 0.73 (mean ± SD) in CTD without thyroiditis  and lower birth weight: 2,790.71 g ± 257.17 SD in CTD with thyroiditis versus 3,019.33 g ± 305.48 g in CTD without thyroiditis . In SLE and APS thyroiditis did not appear to influence pregnancy outcome. However, we suggest investigating anti-thyroid antibodies in all autoimmune diseases with special attention to pregnant women with thyroiditis and CTD.Few data are available evaluating obstetrical outcome when thyroiditis coexist with autoimmune diseases. Objectives of our study were: 1) To assess the prevalence of thyroiditis in pregnant women with autoimmune diseases; 2) To evaluate the effects on pregnancy outcome when different autoimmune diseases are associated with thyroiditis. Two groups of pregnant women were analysed: a study group of pregnant women with autoimmune diseases ( During pregnancy, the maternal thyroid gland faces several metabolic, hemodynamic, and immunologic changes . In partIn fact, autoimmune thyroid disorders are frequently associated with other organ and non-organ-specific autoimmune diseases, especially in major rheumatic diseases (MRD), such as systemic lupus erythematosus (SLE), antiphospholipid syndrome (APS) and connective tissue diseases (CTD) .Many reports in the last decades documented the deleterious impact that thyroid disease has on pregnancy and postpartum period in terms of spontaneous abortion, prematurity, gestational diabetes, low birth weight  or largeAlthough either thyroid or rheumatic autoimmune disorders have been associated with pregnancy complications, there are scarce data in literature on the effect of the association between the two disorders on pregnancy outcome.The pathogenic mechanisms of this coexistence are not completely defined, but genetics, epigenetics, immune defects, hormonal and environmental factors may play pivotal roles in poly-autoimmunity.The objectives of our study were: 1) To assess the prevalence of autoimmune thyroiditis in pregnant women affected by autoimmune diseases in comparison to that of control pregnant women; 2) To evaluate the effects on pregnancy outcome when different autoimmune diseases are associated with thyroiditis in terms of gestational week at delivery, birth weight and birth weight percentile.n = 268) versus a control group of control pregnant women .A retrospective observational study was performed, analysing two groups of pregnant women, consisted by a study group of pregnant women with autoimmune diseases (The study group comprised 268 women with a confirmed diagnosis of autoimmune disease that were followed in our clinic throughout pregnancy and delivered in our centre from January 2010 to September 2020 (Fondazione Policlinico Universitario A. Gemelli—IRCCS). On the other hand, the control group comprised 1,150 pregnant women that consecutively delivered in our centre from September 2018 to December 2018 that included both physiological and complicated pregnancy .Both in the study group and the control group, the research for thyroid antibodies, anti-TPO and anti-TG, was performed during the pregnancy, despite TSH value. In fact, the research involved either euthyroid pregnant women or women with altered thyroid function. In our series of cases, therapy of hypothyroidism was given/adjusted when necessary.Patients with autoimmune diseases were 268 with an overall amount of 294 pregnancies, because some patients had more than one pregnancy in the described time interval. The study group was very heterogeneous, represented pregnancies with APS, SLE, CTD, arthritis, celiac disease, inflammatory bowel disease, Sjogren syndrome and other autoimmune diseases. Some patients had one or more autoimmune diseases.versus control group), multiple pregnancies and intrauterine fetal deaths were not included in the analysis. Therefore, pregnancies with MRD eligible for the analysis were: SLE n = 36, APS n = 44, and CTD n = 23. The three different disorders considered in MRD were divided according to the positive or negative results for thyroid antibodies.The analysis was subsequently focused on pregnancies of patients suffering for major rheumatic diseases (MRD): SLE, APS and CTD. To compare the pregnancy outcome in the two groups   .In the study group the most common autoimmune disease associated with thyroiditis was celiac disease with a rate of 53% of thyroiditis. APS showed a rate of 35%, CTD a rate of 25%, and SLE had a rate of 24%.versus 38.56 ± 0.73 (mean ± SD) in CTD without thyroiditis  and lower birth weight: 2,790.71 g ± 257.17 SD in CTD with thyroiditis versus 3,019.33 g ± 305.48 g in CTD without thyroiditis , and lower birth weight percentile, although the decrease did not reach a statistically significant difference.The analysis of pregnancy outcome in the MRD group, according to the presence of autoimmune antibodies, revealed the following findings. Autoimmune thyroiditis in patients having CTD showed a detrimental effect on pregnancy outcome, in terms of earlier week of delivery: 37.86 ± 0.90 (mean ± SD) in CTD with thyroiditis No statistically significant difference in pregnancy outcome was reported in the other two groups (SLE and APS) when thyroiditis was associated .per se could impair the pregnancy outcome and fecundity in childbearing women. Up to date, only few data are available evaluating the obstetrical outcome when thyroiditis coexists with autoimmune diseases.Autoimmune diseases could be considered a family of disorders that often coexist in the same subject. The isolated thyroiditis Miscarriage and preterm delivery are the most common obstetric complications in pregnant women with isolated thyroiditis, also in presence of a normal thyroid hormone status . It is sConcerning the impact on pregnancy outcome when thyroiditis is associated with autoimmune disorders, the results of the various studies are conflicting and non-conclusive .Our study is one of the few reports evaluating the impact of thyroid antibody positivity on the pregnancy outcome in women affected by autoimmune diseases. Two major considerations can be done: the first one is that the prevalence of thyroiditis was significantly increased in the autoimmune disease pregnant women (17.54%) respect to that of the control group (5.57%); the second one is that the coexistence of thyroiditis with autoimmune diseases negatively impaired the pregnancy outcome in a specific group of rheumatic disorders such as CTD.Regarding the prevalence of thyroid antibodies in MRD pregnancies, our findings confirm prior reports. In fact, the prevalence of thyroiditis in pregnant patients with SLE was of 24% in our study, very similar to the findings by Stagnaro et al. (23.8%). These authors described also a very high rate of preterm delivery (67%) in women who had thyroid disease, compared with the SLE women that were thyroid disease free (18%) .In women with primary APS and history of recurrent spontaneous abortions, it was described a prevalence of 27% of thyroid antibody positivity . FurtherMoreover, we observed in CTD patients a prevalence of 24% of thyroiditis, although in another study the overall prevalence of either anti-TPO or anti-TG detection was up to 62.3% among CTD patients while it was 8% among controls . FurtherSurprisingly, in our study, the presence of anti-TPO and anti-TG antibodies was related with an increased risk of adverse pregnancy outcome, in terms of a lower week of delivery and a lower birth weight only in patients with CTD. Probably, in cases with SLE and APS the results were compromised by the small sample size. Other weaknesses of our analysis were: the retrospective design of the study; the lack of informations about replacement treatment; and the absence of cases with miscarriages, because we recruited women after the earlier weeks of pregnancy.Future larger studies need to clarify the role of thyroid antibodies in the different autoimmune diseases, and the eventual role of thyroid hormone replacement on pregnancy outcome.Finally, it is well known the EULAR recommendations  are to iIn our opinion, it is important to extend the prenatal screening for thyroid antibodies to all women affected by autoimmune diseases, paying special attention to the patients with CDT. On the other hand, it could be a field of interest to do the screening for autoimmune diseases in women in childbearing age with thyroiditis, particularly in presence of new specific symptoms or rheumatic clinical manifestations.Defining the co-morbidities in high-risk pregnancies, as are women with autoimmune diseases, could better stratify the risk profile of the patient and could lead the clinicians to a tailored management of pregnancy.


Streptococcus and Veillonella. We hypothesize that the two stomatotypes could represent the microbial succession from periodontal health to starting dysbiosis. The old and young periodontally healthy subjects do not cluster separately but a trend of the OM in older subjects to periodontitis is visible. Several bacterial genera were identified to be typically more abundant in older periodontally healthy subjects.The estimation of oral microbiome (OM) taxonomic composition in periodontally healthy individuals can often be biased because the clinically periodontally healthy subjects for evaluation can already experience dysbiosis. Usually, they are included just based on the absence of clinical signs of periodontitis. Additionally, the age of subjects is used to be higher to correspond well with tested groups of patients with chronic periodontitis, a disorder typically associated with aging. However, the dysbiosis of the OM precedes the clinical signs of the disease by many months or even years. The absence of periodontal pockets thus does not necessarily mean also good periodontal health and the obtained image of “healthy OM” can be distorted.To overcome this bias, we taxonomically characterized the OM in almost a hundred young students of dentistry with precise oral hygiene and no signs of periodontal disease. We compared the results with the OM composition of older periodontally healthy individuals and also a group of patients with severe periodontitis (aggressive periodontitis according to former classification system). The clustering analysis revealed not only two compact clearly separated clusters corresponding to each state of health, but also a group of samples forming an overlap between both well-pronounced states. Additionally, in the cluster of periodontally healthy samples, few outliers with atypical OM and two major stomatotypes could be distinguished, differing in the prevalence and relative abundance of two main bacterial genera:  Periodontitis is the sixth most common disease worldwide . The majPorphyromonas gingivalis, Tannerella forsythia, and Treponema denticola , 17 periodontally healthy people older than 40 years , and 45 patients with severe (former aggressive) periodontitis . All subjects live in Czech Republic, but besides being students of the same university in HY group, they have no other general relation among each other in terms of living area, employment or any similar parameter. All subjects were examined by a single experienced periodontologist. To be included in the HY or HO group the subjects were required to have no periodontal pocket on probing depth >3 mm, in AP group the subjects had at least two periodontal pockets on probing depth >5mm. The subjects had not received any antibiotic treatment or periodontal therapy in the three months before the beginning of the study. The HO and AP subjects were obtained within the study approved by the Ethics Committee of the First Faculty of Medicine of Charles University and General University Hospital in Prague as a part of project No. 17-30753A of the Czech Health Research Council and besides periodontitis in the AP group, they were of good general health, the sampling of HY subjects was approved within a project No. 486417 from the Grant Agency of Charles University. All subjects involved in the study signed the informed written consent.Samples from 153 subjects were included and analyzed in this study  were taken from the deepest periodontal pocket. The healthy subjects fulfilled the criteria of periodontal health, as defined by Caton et al., , i.e. noAdditional information on the tooth sampled  and other relevant conditions including health state, smoking status, pregnancy, nationality or specific diet are listed in the 2O. Cycling conditions were 94°C for 5 min; 35 cycles of 94°C for 1 min, 58°C for 50 s, 72°C for 30 s, followed by 72°C for 10 min. Pooled PCR products were purified after the electrophoretic separation from the agarose gel using the Wizard SV Gel and PCR Clean-Up System . 3 µl of isolated DNA were used as a template in the second PCR in reaction containing 2 µl of each tagged primer and 25 µl of Plain Combi PP Master Mix enriched by Phusion DNA polymerase and 18 µl of H2O. Cycling conditions were the same except that cycle number was 10 and number of independent reactions per sample was two. PCR products were separated by electrophoresis and purified using the Wizard SV Gel and PCR Clean-Up System and then concentrated into the volume of 16 µl using the MinElute PCR Purification Kit . The concentration of DNA was measured by Qubit 2.0 Fluorometer using dsDNA BR Assay Kit . The purified solutions of tagged amplicons from different samples were mixed in equimolar concentrations, the amplicon library was constructed using TruSeq DNA Library Preparation Kit v2  and sequenced by Illumina MiSeq platform .The DNA was extracted using DNeasy Blood&Tissue kit  according to the modified manufacturer’s instructions  and stored in -20°C. Isolated DNA was used as a template for PCR 16S rDNA amplification. The universal primers 530f (GTGCCAGCMGCNGCGG)  and 907RThe amplicon raw sequencing data were processed using the pipeline SEED 2.0.3 . The paiThe Weighted Jaccard Similarity clustering analysis Figure 1post hoc test was employed to assess the statistical significance of individual taxa relative abundance difference among groups identified by multivariate clustering analysis ; for HY/HO and HO/HP p was > 0.05.The sequencing statistics is summarized in the All 153 samples were clustered using Weighted Jaccard Similarity analysis, index value 0.3 Figure 1Fusobacterium nucleatum, CT8 Porphyromonas pasteri/P. catoniae, HMT775 Capnocytophaga sputigena, HMT329 Capnocytophaga leadbetteri and CT51 Capnocytophaga granulosa, HMT311 Prevotella oris, CT53 Tannerella sp. and HMT623 Campylobacter gracilis.In the health-associated cluster, the most abundant and prevalent taxa are CT2 Streptococcus mitis/S. oralis, CT43 Streptococcus gordonii/S. sanguinis, CT6 Veillonella dispar/V. parvula, CT25 Neisseria flava/N. mucosa, CT27 Neisseria subflava, HMT14 Neisseria oralis, HMT718 Haemophilus parainfluenzae, CT23 Haemophilus haemolyticus, CT24 Haemophilus sputorum, CT10 Prevotella histicola, CT37 Gemella morbillorum, CT48 Rothia dentocariosa, HMT22 Lautropia mirabilis, HMT37 Stenotrophomonas nitritireducens, and CT13 Aggregatibacter aphrophilus Table 1.Fretibacteria CT12, and HMT 363, and Filifactor alocis HMT 539) are slightly lower when compared to the AP group. On the other hand, the average relative abundance of Fusobacterium nucleatum is higher in these six samples. The taxa typical for periodontal health  are almost absent exhibiting the average relative abundance values comparable to the AP group. Even though the 6 HO individuals did not exhibit any clinical signs of the disease, their OM taxonomic composition indicates a high risk of periodontitis development in the future.Even though no obvious age-dependent clustering pattern was observed, still 35% of samples from HO group (6 out of 17), clustered together with AP or in close proximity  and Cluster 2 . This distribution corresponds well also with the main taxa driving the PCA distribution , and three samples localized within the health-associated cluster in  disease . The thr disease . This, h disease . ConsequAggregatibacter actinomycetemcomitans in our AP group , because for a long time, this taxon has been typically associated with periodontitis, mainly with its severe  form  periodontitis in question.The most abundant and prevalent taxa in health and periodontitis-associated cluster are consistent with previously published data . Slightlon) form . NeverthFusobacterium, Porphyromonas or Capnocytophaga), which are supposed to act as later colonizers, facilitating further colonization by “true periopathogens” of red complex . The genus Porphyromonas is quite unique because some Porphyromonas species are frequently associated with oral health , HMT329 Capnocytophaga leadbetteri (1.91%), and CT51 Capnocytophaga granulosa (1.15%). It is not a highly abundant genus but its relative abundance in healthy samples is remarkably lower and in periodontitis, it almost disappears. This finding is in good agreement with Pudakalkatti et al., who described Capnocytophaga species to be the most prevalent in gingivitis  rather than in healthy periodontium and periodontitis , a taxon previously proved to co-aggregate with P. gingivalis and thus to promote the colonization of the gingiva by P. gingivalis in early stage of biofilm formation  associated with periodontitis . In transient state, it increased above 1% and also the prevalence was remarkably higher (89% compared to 43% and 64% in both border states). This finding corresponds well with previous association of C. gracilis with shallow periodontal pockets rather than the deeper ones , no considerable differences in the OM composition were noted with regards to dental caries or periodontitis, between younger and elderly healthy populations (Belibasakis in his recent review summarized the knowledge concerning the OM composition changes in relation to the aging  showing ulations .vs. 19-39 years with average 23 in HY). Generally, these changes could be summarized as an aging-related gradual decrease of relative abundance of health-associated taxa and an increase of taxa associated with the transient state. The most remarkable is the increasing relative abundance of CT3 F. nucleatum and Capnocytophaga species and decreasing relative abundance of health-associated Neisseria species, Lautropia mirabilis, Prevotella histicola or Gemella morbillorum , but according to the P. pasteri/P. catoniae belong to the so-called POTG  group of microorganisms   . This ab unusual . It has nditions . The preNevertheless, it is not only the taxonomic composition of the OM but the overall metabolic activity in the oral habitat including the host response to microbial production, which are the critical factors distinguishing between oral health and dysbiosis resulting in any type of oral pathogenesis. In the majority (~ 90%) of samples, the OM taxonomic composition corresponds well to the state of health and can serve as a fast diagnostic tool, however, still, there are individuals with atypical OM taxonomic composition, the atypical metabolic activity of typical OM or unusual immune reaction toward usual OM – in all these cases, the evaluation of proteome and/or metabolome could provide a more accurate image.https://www.ncbi.nlm.nih.gov/bioproject, PRJNA670573.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The studies involving human participants were reviewed and approved by Ethics Committee of the First Faculty of Medicine of Charles University and General University Hospital in Prague as a part of project No. 17-30753A of the Czech Health Research Council. The patients/participants provided their written informed consent to participate in this study.JJ and LN postulated the hypothesis and designed the experiments. ML participated on experiment design, evaluated sequencing results and prepared first draft of the manuscript. JM diagnosed the patients and performed sampling. TJ assisted in sampling and performed DNA isolation and primary PCR. BT further processed samples for sequencing and participated on evaluation of sequencing results. OH did the clustering analysis and participated on evaluation of results and figure preparation. LN participated on the evaluation of results and finalized manuscript. All authors contributed to the article and approved the submitted version.This study was supported by grant 486417 from the Grant Agency of Charles University and project 17-30753A from Czech health research council.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.