Mycobacterium marinum infection of zebrafish. We found that PDIM's methyl-branched lipid tails enabled it to spread into host epithelial membranes to prevent immune activation. Additionally, PDIM’s affinity for cholesterol promoted this phenotype; treatment of zebrafish with statins, cholesterol synthesis inhibitors, decreased spreading and provided protection from infection. This work establishes that interactions between host and pathogen lipids influence mycobacterial infectivity and suggests the use of statins as tuberculosis preventive therapy by inhibiting PDIM spread.Several virulence lipids populate the outer cell wall of pathogenic mycobacteria. Phthiocerol dimycocerosate (PDIM), one of the most abundant outer membrane lipids, plays important roles in both defending against host antimicrobial programs and in evading these programs altogether. Immediately following infection, mycobacteria rely on PDIM to evade Myd88-dependent recruitment of microbicidal monocytes which can clear infection. To circumvent the limitations in using genetics to understand virulence lipids, we developed a chemical approach to track PDIM during  Mycobacterium tuberculosis, the causative pathogen of the pulmonary disease tuberculosis (TB), is estimated to have evolved within the confines of the human lung for millennia  is required to evade this antibacterial response  signaling . Screeniresponse . PDIM alresponse  and in presponse . HoweverTo accomplish mechanistic studies of virulence lipids, we and others developed metabolic labeling strategies where unnatural metabolic precursors are fed to growing bacteria . The unnHere, we have developed comparable chemical tools to monitor PDIM’s distribution during infection. We found that the first step in PDIM-mediated pathogenesis is to spread into epithelial cells in order to prevent the recruitment of microbicidal monocytes. Structure function analysis revealed that PDIM’s methyl-branched fatty acids increased lipid mobility and promoted spread. Spreading was also dependent on the lipid content of host membranes. Administration of the cholesterol lowering drug, atorvastatin (Lipitor), led to a decrease in PDIM spreading, and subsequent resistance to mycobacterial infection. Our findings provide a mechanistic explanation for the association of statin use with a decrease in TB incidence  and suppM. tuberculosis and M. bovis, we validated that petroleum ether extraction did not affect the growth of M. marinum in culture  and localization to the mycomembrane (∆mmpL7) trigger TLR/Myd88-dependent immune responses  or extracted and recoated with their native lipids or the lipids from the other strain . DIM-488 spread away from bacteria into infected macrophage membranes (Tg(mfap4:tdTomato) whose macrophages express the fluorescent protein tdTomato  which lack EsxA , which has a red-fluorescent vascular endothelium  of metabolically labeled trehalose monomycolate (TMM)  . We confrehalose  followedrehalose . When werehalose . Thus, PIf increased membrane fluidity and lipid mobility promote membrane mixing, then TMM should not be able to spread into host cells as efficiently as PDIM does. Indeed, we found that TMM-488 failed to spread onto epithelial cells at 2 hpi . Only whTo determine if there is a causal relationship between mobility and spreading, we used FRAP to identify conditions that decreased PDIM’s recovery after photobleaching. Neither heat-killing nor mild chemical fixation ) significantly reduced DIM-488’s recovery . HoweverTo further test if PDIM’s mobility promotes its spreading, we sought to take advantage of our ability to alter PDIM’s structure. Lipid structure is known to modulate lipid mobility and membrane fluidity . SaturatM. marinum must be alive in order to stimulate resident macrophages to express CCL2 to recruit permissive monocytes , heat-killed, PFA treated, or PFA+GA treated and then determined the burdens of the red-fluorescent wildtype M. marinum at 3 dpi. Only co-infection with PFA+GA treated bacteria caused attenuation of wildtype bacteria  was found to be host cholesterol M. tuberculosis grown in culture can sequester cholesterol to its outer mycomembrane, a process dependent on mycomembrane lipids  which resulted in an eight-fold decrease in cholesterol . This waM. marinum established infection at a reduced frequency  and AFDye 647 DBCO  are suitable alternatives to the DIBO fluorophores. The DBCO dyes can be used with the same concentrations and labeling protocols presented in this manuscript for the DIBO dyes (Unpublished data).All reactions were performed in dry standard glassware fitted with rubber septa under an inert atmosphere of nitrogen unless otherwise stated. Preparative thin-layer chromatography (TLC) was performed with Millipore’s 1 mm and 0.2 mm silica gel 60 pre-coated glass plates. Analytical TLC was used for reaction monitoring and product detection using pre-coated glass plates covered with 0.20 mm silica gel with fluorescent indicator; visualized by UV light and 10% CuSO4 in 1.3M phosphoric acid in water. Reagents were purchased in reagent grade from commercial suppliers and used as received, unless otherwise described. Anhydrous dichloromethane (DCM) was prepared by passing the solvent through an activated alumina column.1H NMR) and proton-decoupled carbon-13 (13C {1H} NMR) nuclear magnetic resonance spectra (3: 7.26 [CHCl3]) and carbon (CDCl3: 77.16) resonances of the NMR solvent. Data are represented as follows: chemical shift, multiplicity , coupling constants in Hertz (Hz), integration. Mass spectra were obtained on a Bruker Microflex MALDI-TOF by mixing 0.5 µl of 1 mg/ml sample in chloroform with 0.5 µl of 10 mg/ml 2,5-dihydroxybenzoic acid before spotting onto a 96-well MALDI plate.Proton  δ 4.93–4.87 , 3.32 , 2.87–2.83 , 2.57–2.51 , 1.90–0.81 . 13C NMR  δ 176.69, 176.63, 86.78, 86.73, 77.41, 77.37, 77.16, 76.90, 70.72, 70.67, 57.51, 45.56, 45.51, 41.41, 41.33, 38.56, 37.85, 37.73, 37.09, 36.77, 34.91, 34.18, 34.07, 32.83, 32.08, 30.24, 29.96, 29.87, 29.82, 29.75, 29.62, 29.53, 28.34, 27.64, 27.36, 27.25, 27.16, 27.12, 25.74, 25.35, 22.85, 22.74, 22.43, 20.81, 20.57, 20.52, 20.37, 20.27, 18.67, 18.58, 18.53, 14.80, 14.75, 14.28, 14.22, 10.26, 1.16. MALDI-TOF for: C82H162O5 Calc’d [M+Na+]=1250.23; found 1250.41. C84H166O5 Calc’d [M+Na+]=1278.26, found 1278.40. C86H170O5 Calc’d [M+Na+]=1306.29, found 1306.55.Wildtype 1H NMR  δ 5.06–4.86 , 3.30–3.21 , 2.58–2.50  2.1–0.81 . 13C NMR  δ 176.73, 176.63, 77.41, 77.36, 77.16, 76.91, 71.08, 70.74, 59.86, 45.56, 41.96, 41.45, 41.35, 39.27, 37.84, 37.73, 37.24, 37.08, 37.01, 35.28, 34.82, 34.18, 32.08, 30.32, 30.24, 30.22, 30.04, 29.96, 29.87, 29.82, 29.75, 29.72, 29.67, 29.62, 29.52, 29.39, 28.40, 28.34, 27.72, 27.36, 27.25, 27.18, 27.16, 26.20, 25.35, 25.27, 25.23, 22.85, 21.28, 21.13, 20.35, 20.32, 20.27, 18.73, 18.66, 18.58, 18.53, 14.56, 14.29, 13.87. MALDI-TOF for: C81H159N3O4 Calc’d [M+Na+]=1261.22; found 1260.92. C83H163N3O4 Calc’d [M+Na+]=1289.25, found 1289.02. C85H167N3O4 Calc’d [M+NH4+]=1312.33, found 1312.37.To PDIM  was added 0.3 ml of DCM, the mixture was stirred and iodotrimethylsilane  was added and allowed to react for 12 hr at 23°C. The reaction was concentrated under reduced pressure. NaN3  was added followed by 0.3 ml of anhydrous dimethylformamide (DMF) and stirred for 12 hr at 23°C. The solvent was removed under reduced pressure and the product was purified using preparative TLC in 98:2 petroleum ether:ethyl acetate Rf = 0.5 as a white wax . 1H NMR  and 13C NMR  matched previously reported spectra. ESI HRMS for: C28H58O3 Calc’d [M+H+]=443.4464; found 443.4449. C29H60O3 Calc’d [M+H+]=457.4621; found 457.4616. C30H62O3 Calc’d [M+H+]=471.4777, found 471.4762.To dried PDIM  was added 0.5 ml of 40% tetrabutylammonium hydroxide in water, the mixture was sealed, aggressively stirred, and allowed to react for 48 hr at 120°C. Reaction was cooled to 23°C and 4M HCL was added dropwise until pH was <3. The reaction was extracted with DCM and washed once with water. The solvent was removed under reduced pressure and phthiocerol was purified using preparative TLC in 80:20 petroleum ether:ethyl acetate Rf = 0.4. .1H NMR  δ 4.94–4.87 , 3.33 , 2.88–2.83 , 2.30–2.25 , 1.75–0.79 . 13C NMR  δ 173.42, 86.66, 77.27, 77.02, 76.76, 70.97, 57.40, 38.45, 34.84, 34.67, 34.13, 34.09, 32.62, 31.94, 29.73, 29.68, 29.55, 29.47, 29.38, 29.34, 29.23, 27.44, 25.57, 25.17, 25.10, 22.70, 22.37, 14.76, 14.12, 10.10. MALDI-TOF for: C76H150O5 Calc’d [M+Na+]=1166.14, found 1166.14. C77H152O5 Calc’d [M+Na+]=1180.15, found 1180.24. C78H154O5 Calc’d [M+Na+]=1194.17; found 1194.25.To phthiocerol  was added lignoceric acid , N,N’-dicyclohexylcarbodiimide , 4-Dimethylaminopyridine , and 2.3 ml of DCM. The reaction was stirred and allowed to react for 16 hr at 23°C. The solvent was removed under reduced pressure and the product was purified using preparative TLC in 98:2 petroleum ether:ethyl acetate Rf = 0.4. . 3  was added followed by 0.3 ml of anhydrous dimethylformamide (DMF) and stirred for 12 hr at 23°C. The solvent was removed under reduced pressure and the product was purified using preparative TLC in 98:2 petroleum ether:ethyl acetate Rf = 0.5. . 1H NMR  δ 5.08–5.00 , 3.32–3.20 , 3.12–3.04 , 2.33–2.25 , 1.80–0.73 . 13C NMR  δ 173.41, 77.27, 77.02, 76.76, 71.16, 70.05, 59.72, 39.09, 36.92, 35.12, 34.70, 34.64, 32.34, 31.94, 29.72, 29.67, 29.58, 29.53, 29.42, 29.38, 29.33, 29.23, 26.05, 25.15, 24.02, 22.70, 21.15, 16.10, 14.13, 11.06, 1.03. ESI C75H148NO4 calc’d =1166.10, found 1166.05.To PDIF  was added 0.3 ml of DCM, the mixture was stirred and TMSI  was added and allowed to react for 3 hr at 23°C. The reaction was concentrated under reduced pressure. NaNTg(mfap4:tdTomato) , and dTomato)  lines weM. marinum strain M (ATCC BAA-535) and ∆mmpL7 mutants (msp12 promoter were grown under hygromycin (Thermo-Fisher) selection in 7H9 Middlebrook’s medium (Difco) supplemented with 10% OADC (Fisher), 0.2% glycerol, and 0.05% Tween-80 (Sigma). Where noted bacteria were also grown in glycerol-alanine-salts (GAS) medium, recipe (%weight/volume) in 18 mM sodium hydroxide in Milli-Q water pH 6.6 +/- 0.05% Tween-80: 0.03% BactoCasitone (BD Science), 0.005% ferric ammonium citrate (Sigma), 0.4% potassium phosphate dibasic anhydrous (VWR), 0.2% citric acid, anhydrous (VWR), 0.1% L-alanine (Sigma), 0.12% magnesium chloride, heptahydrate (VWR), 0.06% potassium sulfate (VWR), 0.2% ammonium chloride (VWR), and 1% glycerol. To prepare heat-killed M. marinum, bacteria were incubated at 80°C for 20 min. To prepare fixed bacteria, bacteria were incubated in described concentrations of glutaraldehyde (Sigma) and/or paraformaldehyde (Thermo-Fisher) for 1 hr at 23°C, followed by three washes with PBS prior to experimental use. mutants  expressiM. marinum at a multiplicity of infection (MOI) of 2 in growth media. Infection was allowed to progress for 6 hr prior to washing twice with PBS and replacing with growth media. THP-1s were then incubated for 24 hr at 33°C and 5% CO2 prior to experimental end point. One day prior to infection, A549s were plated on 8-well chamber Nunc Lab-Tek II chamber slides (Thermo-Fisher) at a density of 50,000 cells per well in growth media and incubated at 37°C and 5% CO2. For cholesterol labeling, BODIPY-cholesterol  was added at 3 µg/ml one day prior to infection and cells were incubated overnight at 37°C and 5% CO2. Cells were then washed three times with PBS prior to infection. Day of infection cells were moved to 33°C and 5% CO2 for 3 hr, and then were infected with M. marinum at a MOI of 5 and incubated at 33°C and 5% CO2 for 24 hr. Infected THP-1s and A549s were then imaged as described below.All cell lines were from ATCC and were used at passages < 10. Cell lines were authenticated by the supplier and were verified to be mycoplasma negative by Lonza MycoAlert Mycoplasma Detection Assay. Cells were grown in T75 flasks (Thermo-Fisher) and maintained at 37°C and 5% CO2. THP-1 were grown in RPMI supplemented with 10% fetal bovine serum (FBS). A549 were grown in DMEM supplemented with 10% FBS. Two days prior to infection, THP-1s were plated on 8-well Nunc Lab-Tek II chamber slides (Thermo-Fisher) at a density of 150,000 cells per well with 100 nM PMA  in growth media and incubated at 37°C and 5% CO2 for 24 hr. One day prior to infection, the media on THP-1s was replaced with fresh growth media and the cells were incubated at 33°C and 5% CO2 for 24 hr, cells were then infected with M. marinum were grown in GAS medium plus Tween-80 to an OD600 of 1.2. Bacteria were pelleted in a glass 50 ml conical tubes (Fisher) of known weight, frozen and lyophilized. The dry bacteria in 50 ml conical tubes were then weighed and the dry bacterial weight was calculated. 25 ml of petroleum ether were then added to the bacteria, and the conical tube was capped with a PTFE lined lid (Sigma) and the suspension was vortexed for 3 min. An additional 25 ml of petroleum ether was added and the sample was centrifuged for 3 min at 1000xg at 4°C. The extract was then saved or discarded depending on the downstream experimental applications and the bacteria were extracted once more as above. For total lipid extractions (not used for recoating), bacteria were treated with 1:1 chloroform:methanol for 12 hr at 60°C. Extracts were filtered, back extracted with water to remove water-soluble contaminants, dried under reduced pressure and used for downstream experiments.1 liter of Prior to extractions the following calculation was used to determine the amount of lipids to add back to each pellet.(23 mg lipid/gram dry bacteria)*(weight in grams of dry bacteria)/(0.75 recoating efficiency).Where 23 mg of lipid per gram of dry bacteria is the experimentally determined average amount of lipid removed during petroleum ether extraction of bacteria grown in these conditions. . If recoFor more thorough details, as well as rationale and explanation of the following protocol see E. coli expressing the tret gene from Thermoproteus tenax  were streaked onto a Lysogeny broth (LB) agar plate supplemented with 100 µg/ml ampicillin and incubated at 37°C for 24 hr. A single colony was picked and used to inoculate 5 ml of LB liquid medium containing 100 µg/ml ampicillin. The starter culture was placed in a shaking incubator (175 rpm) at 37°C for 16 hr. The starter culture was then transferred to a 1 L solution of sterilized Terrific broth (TB) supplemented with 100 µg/ml ampicillin in a sterilized 2 L Fernbach culture flask. The flask was shaken (175 rpm) at 37°C. When the absorbance at 600 nm reached between 0.6 and 0.9 , TreT expression was induced by adding 1 ml of 1 M arabinose solution in sterile water . The flask was again shaken (175 rpm) at 37°C for another 20 hr. The culture was then transferred to a polypropylene bottle and pelleted for 15 min at 4000 x g at 4°C. The supernatant was discarded, and the pellet was suspended in 45 ml of lysis buffer  and run through a homogenizer five times utilizing ice to keep the solution cool. Once homogenized, the lysate was clarified by centrifugation pelleting for 20 min at 21,000 x g at 4°C and filtering through a 0.45 µm Teflon syringe filter. To the clarified lysate  was then added 5 ml of pre-washed (lysis buffer) Ni-NTA resin slurry (Qiagen). The suspension was mixed on an orbital shaker for 60–90 min at 4°C and then transferred to a glass column (BIO-RAD EconoColumn). Non-His-Tagged proteins were eluted with lysis buffer until the absorbance at 280 nm matched background levels utilizing 50–100 ml of lysis buffer. His-tagged TreT was eluted with elution buffer  in multiple 2.5 ml increments, until protein elution was determined complete by absorbance at 280 nm. Buffer exchange to a storage/reaction buffer  was performed with a desalting column  and the protein was transferred to a conical tube and diluted to 1 mg/ml as determined by absorbance at 280 nm for long-term storage. Storage of the enzyme at 4°C and at this concentration yields active protein that does not have significant losses in activity even after 12 months of storage under these conditions.TreT was expressed and purified utilizing a similar method as previously reported . Top10 E2 , and 300 ml of reaction buffer . Then, 300 µl of TreT solution in storage/reaction buffer was added  and the reaction vessel was closed and mixed gently by inverting the tube. The reaction was heated to 70°C for 60–90 min with shaking at 400 rpm and then cooled on ice before further manipulation. The reaction contents were transferred to a pre-washed Amicon centrifugal filter with a nominal molecular weight limit (NWML) of 10 kDa. The filter was washed with DI water (2 × 1 ml) to facilitate maximal recovery. The upper chamber was discarded and 1 g of prewashed mixed-bed ion-exchange resin (DOWEX) was added to the filtrate and the slurry was equilibrated for 60–90 min. The suspension was filtered, and the resin was rinsed with 3–5 ml of DI water. Analysis by TLC (5:3:2 n-butanol: ethanol: DI water) and/or LC-MS equipped with a Supelco aminopropyl column   indicated full and complete conversion of 6-azido-6-deoxy-glucose to 6-azido-6-deoxy-trehalose with high purity as determined by nuclear magnetic resonance (NMR), which matched previously reported spectra.To a 1.5 ml Eppendorf tube was added 200 µl of a 100 mM solution of 6-azido-6-deoxy-glucose , 100 µl of a freshly-prepared 400 mM solution of UDP-glucose , 100 µl of a 200 mM solution of MgClM. marinum with 6-TreAz, the reaction was performed as above but 6-TreAz was not isolated prior to labeling. Upon reaction completion, the reaction was cooled on ice to bring the vessel back to ambient temperature. The 10 mM stock of 6-TreAz was then added to cultures of M. marinum to a final concentration of 50 µM.To facilitate easier production and labeling of M. marinum were washed twice with PBS and resuspended in 0.1 M sodium acetate (Sigma), pH 5.5 containing 1 mM sodium periodate (Sigma). Following a 20 min incubation at 4°C with gentle rotation, 0.1 mM glycerol was added to stop the reaction. Cultures were washed three times with PBS and then subjected to single cell preparation. Following single cell preparation, pellets were transferred to a 96-well plate and then incubated with PBS containing 1 mM Alexa-647 hydroxylamine (Thermo-Fisher). Following a 2 hr incubation at 23°C, the cultures were washed five times with PBS and twice with prep media.Surface-exposed terminal oxidizable carbohydrates were labeled with hydroxylamine following periodate oxidation . LyophilFollowing recoating or metabolic labeling, bacteria were treated for single cell preparation. Bacteria were then transferred to 96-well v-bottom dishes and were washed twice with PBS using centrifugation at 3000xg for 3 min between washing. Bacteria were then stained with either 5 µM DIBO-488 (Thermo-Fisher), or 30 µM DIBO-647 (Thermo-Fisher) in 200 µl PBS for 90 min at 23°C protected from light. Bacteria were then washed five times in PBS followed by two washes in prep media. Bacteria were then aliquoted and stored at −20°C for future use. Staining efficiency was evaluated by flow cytometry on a BD-Accuri C6 Plus and analysis was performed using the FlowJo software package. Staining was also evaluated by microscopy with a 60x oil-immersion Plan Apo 1.4 NA objective on the Nikon A1R confocal microscope.GTTAAACACTGACCCTGTGGATCAT3’ (http://clodronateliposomes.org) were diluted 1:10 in PBS and injected into 2-dpf-old larvae in ~10 nl via the caudal vein.To generate Myd88 knockdown zebrafish larvae, the Myd88 morpholino 5’GATCAT3’  was diluLarvae were embedded in 1.5% low melting point agarose (Thermo-Fisher) and a series of z stack images with a 2 µm step size was generated through the infected HBV. For infected THP-1 and A549 cells, a series of z stack images with a 1 µm step size was generated. Images were captured using the galvo scanner (laser scanner) of the Nikon A1R confocal microscope with a 20x Plan Apo 0.75 NA objective. Higher resolution images were generated using a 40x water-immersion Apo 1.15 NA objective. Bacterial burdens were determined by using the 3D surface-rendering feature of Imaris (Bitplane Scientific Software). Spread lipid images were generated by subtracting the bacterial surface from the lipid channel. 3D surface-rendering was then done in Imaris on both the total lipid and spread lipid images to generate a percent spread value .M. marinum at the dose reported in the figure legends. At 3 hr post infection, the number of total myeloid cells in the HBV was quantified using differential interference contrast microscopy using a 20x Plan Fluor 0.75 NA objective on Nikon’s Ti eclipse inverted microscope. For quantification of monocytes  (Around 40–50 tdTomato expressing wildtype fection) .3 days post-fertilization larvae were infected via the hindbrain ventricle with an average of 0.8 bacteria per injection. Fish harboring 1–3 bacteria were identified at 5 hr post infection by confocal microscopy. These infected fish were then evaluated at three dpi and were scored as infected or uninfected, based on the presence or absence of fluorescent bacteria .Petroleum ether extracts were dissolved by adding 30 µl of DMSO to 1 mg of lipids. 10 µl of 4x loading buffer (Licor) was then added to each sample and samples were heated at 95°C for 5 min. Samples were then separated on a 4–12% XT Bis-Tris Protein Gel (BioRad), in XT MES running buffer (BioRad) at 200 volts for 40 min. The gel was then stained with Colloidal Coomassie Brilliant Blue for 20 hr at room temperature with shaking. The gel was rinsed with water and then imaged.FRAP experiments were performed using the galvo scanner (laser scanner) of the Nikon A1R confocal microscope with a 60x oil-immersion Plan Apo 1.4 NA objective. Photobleaching was performed with the 405 nm laser for 200 ms on a region of interest (ROI) encompassing ~1 μm from one pole of a single bacteria. A series of images was taken every second over the course of 31 s, one prior to bleaching. Labeled cells were mounted on a slide and coverslip in 0.75% low melting agarose. NIS Elements software (Nikon) was used to analyze the FRAP data to extract the fluorescence recovery kinetics. Briefly, the first image before photobleaching was used to generate an ROI for the entire cell and a second ROI was generated in the photobleached area. Total fluorescence intensities in both the whole cell area and the bleached area were extracted and normalized to correct for photobleaching of the dyes due to acquisition. The normalized fluorescence intensities of the bleached area were then fitted to a non-linear regression with a one-phase association, with the plateau values from each sample plotted to represent the mobile fraction .mmpL7 M. marinum expressing wasabi fluorescent protein were grown in 7H9 medium supplemented with 10% OADC, 0.2% glycerol with or without 0.005% alkyne-cholesterol or azido-PE for 48 hr. Bacteria were then washed 3x with PBS prior to detection with fluorescent probes. For azido-PE, we followed the Copper-free click chemistry of M. marinum protocol above using DIBO-647. For alkyne-cholesterol we performed a copper-click reaction with AlexaFluor-647 Azide. For copper click: 400 µM BTTP (Click Chemistry Tools) and 200 µM copper sulfate (Sigma) were dissolved in PBS and allowed to complex for 20 min. 30 µM AlexaFluor-647 Azide (Thermo-Fisher) and 1.2 mM sodium ascorbate (Sigma) were then added to the solution. Bacterial pellets in 96-well v-bottom plates were resuspended in 50 µl of the solution and were incubated at 23°C protected from light for 45 min. Bacteria were then washed 5x in PBS prior to imaging on a Nikon A1R confocal microscope with a 60x oil-immersion Plan Apo 1.4 NA objective. Nikon elements software was used to determine fluorescent intensities of wasabi and cholesterol signals calculated from line profiles drawn perpendicular to bacterial membranes at least 0.5 µm from either pole.Wildtype or ∆M. marinum at an MOI of 5 for 24 hr at 33°C followed by imaging on a Nikon A1R confocal microscope with a 20x Plan Apo 0.75 NA objective. 2 µm z-stacks were generated through the infected cells. Azido-DIM spreading was calculated similar to above . Cells plated on 24-well plates were rescued in DMEM + 10% FBS for 3 hr at 33°C. Cells were then harvested for quantification of cholesterol levels.A549 cells were seeded at 50,000 cells per 8-well Nunc Lab-Tek II chambered coverglass or at 100,000 cells per well in a 24 well plate. Cells were incubated at 37°C for 48 hr. Cells were washed 1x in PBS followed by treatment with 10 mM methyl-ß cyclodextrin (Sigma), 1 mM water-soluble cholesterol (Sigma), or a combination of 10 mM methyl-ß cyclodextrin and 1 mM water-soluble cholesterol in serum free DMEM media. Cells were treated for 1 hr at 33°C followed by three washes with PBS. Cells plated on chambered coverglass were infected with azido-DIM labeled At 48 hr post-fertilization, 0.5 µM atorvastatin (Sigma), or 10 µM water-soluble cholesterol (Sigma), or both were added to zebrafish water containing 1% DMSO. Control fish were incubated in water with 1% DMSO only. Zebrafish were incubated for 24 hr prior to infection or to cholesterol quantification. Drugs were replenished every 24 hr until experiment endpoint.Cholesterol was quantified using the Total Cholesterol and Cholesterol Ester Colorimetric/Fluorometric Assay Kit (BioVision). For zebrafish, eight larvae were euthanized, transferred to a 1.5 ml Eppendorf tube and excess water was removed. A solution of chloroform:isopropanol:NP-40 (7:11:0.1) was added and the sample was sonicated in a water bath for 1 hr. Samples were then centrifuged at 16,000xg for 10 min and supernatants were transferred to a fresh tube and were allowed to dry in a 60°C water in a chemical fume hood. For A549 epithelial cells, chloroform:isopropanol:NP-40 (7:11:0.1) was added directly to cells in a 24 well plate. Cells were scrapped and solution was transferred to a 1.5 ml Eppendorf tube and was centrifuged at 16,000xg for 10 min and supernatants were transferred to a fresh tube and were allowed to dry in a 60°C water in a chemical fume hood. Dried lipids were then subjected to manufacturer’s protocol and total cholesterol concentrations were determined by fluorescence on a SpectraMax i3x plate reader (Molecular Devices).n value is given and not represented graphically in the figure, n represents the number of zebrafish used for each experimental group : When appropriate D'Agostino-Pearson normality test was done to determine if all of the groups in a particular data set were of a gaussian distribution which then guided the subsequent statistical test performed . Where tal group . In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:M. marinum in the zebrafish. Herein, the authors describe an innovative chemical approach to track PDIM in vitro and in vivo. The authors find that bacterial PDIM spreads into host epithelial cell membranes to prevent immune activation. The authors show that host cholesterol promotes the spreading of PDIM in the membrane. This work establishes that interactions between host and pathogen lipids influence mycobacterial pathogenesis.This manuscript builds on earlier work by this group investigating the role of phthiocerol dimycocerosate (PDIM) in the pathogenesis of Decision letter after peer review:eLife. Your article has been reviewed by Dominique Soldati-Favre as the Senior Editor, a Reviewing Editor, and three reviewers. The following individual involved in review of your submission has agreed to reveal their identity: Stefan H Oehlers (Reviewer #3).Thank you for submitting your article "Spreading of a mycobacterial cell surface lipid into host epithelial membranes promotes infectivity" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife". Please let us know if you would like to pursue this option. As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv  along with this decision letter and a formal designation that the manuscript is "in revision at Summary:M. marinum in the zebrafish. The authors reported that PDIM spreads into epithelial cells in order to inhibit TLR signaling and promote infectivity. The study describes an innovative chemical approach to track PDIM in vitro and in vivo, and presents several interesting results supporting their previous work and data from other groups. Major strengths are the authors' unique expertise and resources, especially in chemical modification of bacterial lipids and the zebrafish model of M. marinum infection. However, some of the data require additional controls and further experiments to conclusively support the authors' conclusion. In particular, (1) the conclusions regarding the PDIM/TLR axis require further dissection and consideration of alternative interpretations of the data presented, (2) the conclusions related to the spreading of PDIM into epithelial membranes require more controls to be convincing, and (3) the authors should compare their current results more extensively to previous findings to better illustrate the broader context of the topics addressed.This manuscript builds on earlier work by this group investigating the role of phthiocerol dimycocerosate (PDIM) in the pathogenesis of Essential revisions:marinum and this is the reason for the altered cellular recruitment that the authors observe. The authors suggest PDIM might disrupt TLR rafting into cholesterol rich domains and sensitivity of THP-1 cells to PDIM spreading. Could they combine their fluorescent PDIM tool with traditional TLR pathway analysis tools in THP-1 cells (where these tools presumably work better than intact zebrafish)? It seems likely that TLR dimerization or association with adaptors like MYD88 are likely mechanisms and should be straightforward to test. PDIM + vs – epithelial cells could also be isolated for proteomic or transcriptional analysis to provide some insight into the mechanism of PDIM inhibition of TLR sensitivity. Notably, the authors previous work suggested that PDIM masked TLR-ligands, do they observe the same masking effect with azido-DIM?1) The evidence presented here suggests that TLRs might be inhibited by PDIM spreading in the membrane, however, the evidence provided here do not directly show this. It is possible that the cytokine response to δ mmpL7 mutants are distinct from WT 2) The microscopy is difficult to interpret in a convincing manner. While it is clear that there are DIM-488 foci that are not colocalized with the bacteria, it is not clear that this is in the host cell membrane. Colocalization with a host cell membrane dye would be a more convincing demonstration of this finding. Since it is not clear that the PDIM signal are in host membranes from the data provided, it raises the possibility that the spots are cytosolic lipid droplets. Could these foreign inclusions could be disrupting host cell biology? The proposal that PDIM interaction with cholesterol promotes spreading into epithelial membranes could be supported by labeling membrane cholesterol and examining how azido-DIM is distributed relative to cholesterol.M. marinum to restore native PDIM in the envelope?In addition, the effects of lipid reconstitution on the lipid and proteomic composition of the mycomembrane should be addressed and considered. How did chemical extraction and reconstitution help control the composition of the mycomembrane ? Is the organization of added lipid similar to that of the lipids transported from the cytoplasm? Characterization of the envelope of mycobacteria reconstituted with azido-DIM relative to intact bacteria is important for interpreting the data on the spread of azido-DIM into the epithelial cell. Since some experiments last up to 3 days, what is the stability of the fluorescence signal of azido-DIM during bacterial growth and the time required for M. marinum and mmpL7 mutant, and MmpL7 transports PDIM and PGL in M. marinum, differences in PGL could also be contributing to the phenotypes.In Figure 1, the authors perform lipid-swap experiments with wild-type In Figure 4, the authors propose that PDIM accumulated in "mycomembrane domains", but this term is poorly defined and images of the microdomains are not provided. The authors also state that mycomembrane domains containing PDIM have a uniquely high fluidity that would promote membrane mixing and contribute to PDIM spreading. Another possibility is that the membrane mixing may help PDIM to be transferred to the host membrane, where they could spread according to their diffusion properties. To test this, the authors could take advantage of A549 cells and compare the spread of DIM-499 in cells treated with DIM-499 or put in contact with DIM-499-labeled bacteria. Cell membranes are highly heterogeneous structures with distinct fluidity from one cell type to another. If PDIM can also diffuse in cell membranes, this could explain the difference in the kinetic of azido-DIM spreading between epithelial cells and macrophages.M. marinum WT and mmpL7 mutant influence their capacity to sequester cholesterol. The authors could consider testing another hydrophobic lipid to further underscore this point.For Figure 7, the rationale for the cholesterol study is interesting given that cholesterol is coupled to biosynthesis of methyl-branched lipids such as PDIM  and the role of DIM in macrophage invasion required cholesterol . Figure 7A-D: Can the distinct hydrophobicity between 3) Crucial data is also unnecessarily buried in supplementary figures. Supplemental Figure 3 is important to the interpretation of the results and needs to be more visible as part of a main figure. As there is no display item limit, so consider integrating the "validation" data into Figure 3 or splitting up Figure 3. Finally, the authors should compare their current results more extensively to previous findings to better illustrate the broader context of the topics addressed.eLife. Your revised article has been evaluated by Dominique Soldati-Favre (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Spreading of a mycobacterial cell surface lipid into host epithelial membranes promotes infectivity" for further consideration by The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:The reviewers appreciate the careful attention of the authors to address most of the original comments, which has provided stronger support of their interpretations and conclusion. However, there are a few points that still need attention as detailed here. These comments should be able to be addressed by changes in the text.1) Claims of TLR inhibition (starting in the abstract and throughout the manuscript) need to softened or removed as this is not directly addressed in the manuscript. The authors show effects on monocyte recruitment and that MyD88 deletion affects monocyte recruitment, but the experiments do not directly show PDIM is inhibiting TLRs and really just show that MyD88 is required for monocyte recruitment.M. tuberculosis would provide strong support for their findings.2) The authors should comment on the membrane effects of PDIM in the context of the literature . These recent data obtained with PDIM of 3) Introduction: Could the authors clarify what fluidity means for a lipid? Membrane fluidity and lipid mobility may be more appropriate.M. marinum”: The authors clearly showed that by mixing of extracted lipids with bacteria, the lipids coat M. marinum, but the authors do not demonstrate that the mycomembrane is reconstituted. For instance, what is the localization and orientation of lipids once coated on the bacteria in comparison to native lipids? This can be addressed by more specifically writing "coating of bacteria with extracted lipid" instead of "reconstitution of the mycomembrane".4) Subsection “Chemical Extraction and Reconstitution of the Mycomembrane of 5) subsection “Pre-infection PDIM Reservoirs are Required for Virulence”: The authors should mention and discuss that deletion of mas or mmpL7 also results in defects in PGL synthesis and localization.6) Subsection “PDIM Spreads into 148 Host Macrophage Cell Membranes” and thereafter: The authors should define more clearly the notion of spreading. Did Azido-DIM spread within the same membrane, suggesting a lateral diffusion, and/or propagate to distinct membrane compartments?7) subsection “PDIM’s Fluidity Promotes Spread into Epithelial Cell Membranes”: The authors claimed that PDIM's fluidity correlates with the lipid's ability to spread into host membrane. Another possibility could be that PDIM's ability to diffuse into the bacterial membrane increases the membrane fluidity, which facilitates membrane mixing. The consequence would be the transfer of PDIM to the host membrane, in which the lipids inserted and can diffuse laterally, as previously reported . The effect of PDIM on bacterial membrane fluidity could therefore be an explanation for the transfer of the lipid rather than for its spreading. In contrast, TMMs are less capable of diffusing and getting transferred. TMM are amphiphilic molecules in comparison to the highly hydrophobic PDIM. This strongly suggests a relationship between lipid structure and capacity to get transferred to host membranes. Please discuss this point in the Discussion section.8) The authors should be clear in their conclusions and the Discussion that the data have been obtained with bacteria coated with extracted lipids.9) The additional images provided by the authors are encouraging but there is not sufficient detail about the quantification to understand if this is representative or not. Furthermore, while the authors are showing overlapping signals with the plasma membrane, these images do not prove that PDIM is in the membrane. To show that PDIM is in the membrane, a z stack is required but not provided. Essential revisions:1) The evidence presented here suggests that TLRs might be inhibited by PDIM spreading in the membrane, however, the evidence provided here do not directly show this. It is possible that the cytokine response to δ mmpL7 mutants are distinct from WT marinum and this is the reason for the altered cellular recruitment that the authors observe.mmpL7 M. marinum. From prior work , we know that wildtype bacteria induce the chemokine CCL2 and that expression of the receptor CCR2 on monocytes is required for their ability to be recruited to the hindbrain ventricle. In this setting, TLR-signaling is not required for monocyte recruitment. Alternatively, when bacteria lack PDIM, we know that CCR2 signaling is no longer required to recruit monocytes but now TLR signaling is required. What we didn’t know at the time was when bacteria lack PDIM, where is detection by TLRs occurring. In this study our data suggests that TLR signaling is happening immediately following infection at epithelial cells at the site of infection. Presumably this then leads to the induction of cytokines/chemokines that are distinct from what occurs during wildtype bacterial infection. Identification of which cytokines are functionally responsible for this phenotype is ongoing.We absolutely agree with the reviewers that different cytokine responses are most likely underlying the different cell recruitment phenotypes seen toward wildtype and ∆The authors suggest PDIM might disrupt TLR rafting into cholesterol rich domains and sensitivity of THP-1 cells to PDIM spreading. Could they combine their fluorescent PDIM tool with traditional TLR pathway analysis tools in THP-1 cells (where these tools presumably work better than intact zebrafish)? It seems likely that TLR dimerization or association with adaptors like MYD88 are likely mechanisms and should be straightforward to test. PDIM + vs – epithelial cells could also be isolated for proteomic or transcriptional analysis to provide some insight into the mechanism of PDIM inhibition of TLR sensitivity. Notably, the authors previous work suggested that PDIM masked TLR-ligands, do they observe the same masking effect with azido-DIM?We are currently searching for the most appropriate in-vitro model system to address this hypothesis. Our data in this manuscript demonstrates that PDIM is required to spread into epithelial cells to prevent recruitment of microbicidal monocytes. Because of this, we don’t anticipate data generated in monocyte cell lines to be relevant. Furthermore, previous literature suggests that TLR signaling may not be fully functional in A549 epithelial cells . Nevertheless, we evaluated the ability of A549 epithelial cells to respond to TNFa, LPS (TLR4 agonist), and PAM3CSK4 (TLR2 agonist) using an NFkB luciferase reporter A549 cell line . Following an overnight incubation, the cells clearly respond to TNFa stimulation but do not respond to TLR agonists (See data in 2) The microscopy is difficult to interpret in a convincing manner. While it is clear that there are DIM-488 foci that are not colocalized with the bacteria, it is not clear that this is in the host cell membrane. Colocalization with a host cell membrane dye would be a more convincing demonstration of this finding. Since it is not clear that the PDIM signal are in host membranes from the data provided, it raises the possibility that the spots are cytosolic lipid droplets. Could these foreign inclusions could be disrupting host cell biology? The proposal that PDIM interaction with cholesterol promotes spreading into epithelial membranes could be supported by labeling membrane cholesterol and examining how azido-DIM is distributed relative to cholesterol.dim’s Fluidity Promotes Spread into Epithelial Cell Membranes”). Finally, we labeled A549 epithelial cells with BODIPY-cholesterol and imaged PDIM spreading onto these cells and found PDIM to strongly co-localize with BODIPY-cholesterol .We appreciate the reviewers’ scrutiny with regards to our microscopy data. We have included additional experiments that support our claims that PDIM is spreading into non-macrophage cell membranes. First, we took advantage of the transgenic zebrafish line Tg(flk1:mcherry), whose vascular endothelial cells express the fluorescent protein mcherry. Following intravenous infection with DIM-488 labeled mycobacteria we captured bacteria in contact with endothelial cells with spread DIM-488 co-localizing with the endothelial marker . Additionally, we used the plasma membrane label Alexa-fluor 594 wheat germ agglutinin to label A549 epithelial cells. We then captured DIM-488 having spread and co-localizing with the plasma membrane label following infection ? Is the organization of added lipid similar to that of the lipids transported from the cytoplasm? Characterization of the envelope of mycobacteria reconstituted with azido-DIM relative to intact bacteria is important for interpreting the data on the spread of azido-DIM into the epithelial cell. Since some experiments last up to 3 days, what is the stability of the fluorescence signal of azido-DIM during bacterial growth and the time required for M. marinum to restore native PDIM in the envelope?M. marinum”. To address concerns over the lipid content of the mycomembrane pre and post reconstitution we extracted lipids from untreated or reconstituted bacteria and compared them by TLC. No apparent differences were noticed . We additionally ran proton NMRs of the two crude extracts from control or reconstituted bacteria, the spectra were near identical . To evaluate how extraction may alter proteins on the mycomembrane we first asked if any proteins were removed during petroleum ether extraction. We resuspended 1 mg of lipid extract in 30µl of DMSO and separated the sample by SDS-PAGE followed by staining overnight with Colloidal Coomassie Brilliant Blue. Similar to previous findings  we found no evidence of protein in the petroleum ether extracts , thus we do not anticipate any changes to the mycomembrane protein composition arising following reconstitution. Furthermore, the ability of reconstituted bacteria to survive as well as untreated bacteria following infection further supports this idea. Additionally, we evaluated how long it takes for delipidated bacteria to repopulate their mycomembrane lipids. Immediately following extraction, delipidated bacteria were rescued into complete growth medium. Every 24hrs these bacteria were harvested and re-extracted and the amount of lipids removed was measured. Even after 5 days in culture, bacteria did not fully restore their mycomembrane lipids . Finally, we grew DIM-488 labeled bacteria for several days following reconstitution and found that the intensity of DIM-488 decreased but was still present on bacteria even after 3 days in culture , suggesting that PDIM is not appreciably shed during mycobacterial growth in aqueous medium and that PDIM spreading is a result of interactions with the host.The chemical extraction and reconstitution method allows us to control the lipid content of the outer mycomembrane, as is evidenced by later sections in the paper. As such we have renamed this subsection “Chemical Extraction and Reconstitution of the Mycomembrane of In Figure 1, the authors perform lipid-swap experiments with wild-type M. marinum and mmpL7 mutant, and MmpL7 transports PDIM and PGL in M. marinum, differences in PGL could also be contributing to the phenotypes.The reviewers are correct that phenolic glycolipid (PGL) also depends on MmpL7 for its localization to the mycomembrane. Using mutants defective for PGL synthesis, that retain PDIM on their surface, previous studies found that these phenotypes (decreased growth following hindbrain infection with ~100 bacteria and TLR-dependent monocyte recruitment following hindbrain infection with ~100 bacteria) were not influenced by the absence of PGL . Therefore, we decided to leave this detail of mycobacterial cell wall biology out of the manuscript so as not to overwhelm the readers.In Figure 4, the authors propose that PDIM accumulated in "mycomembrane domains", but this term is poorly defined and images of the microdomains are not provided. The authors also state that mycomembrane domains containing PDIM have a uniquely high fluidity that would promote membrane mixing and contribute to PDIM spreading. Another possibility is that the membrane mixing may help PDIM to be transferred to the host membrane, where they could spread according to their diffusion properties. To test this, the authors could take advantage of A549 cells and compare the spread of DIM-499 in cells treated with DIM-499 or put in contact with DIM-499-labeled bacteria. Cell membranes are highly heterogeneous structures with distinct fluidity from one cell type to another. If PDIM can also diffuse in cell membranes, this could explain the difference in the kinetic of azido-DIM spreading between epithelial cells and macrophages.We agree with the reviewers that our interpretation that PDIM resides in distinct membrane domains is not accurate. We have changed our description to better reflect our data. We also appreciate your inquiry into the mechanism behind PDIM spreading. Your suggestion that membrane mixing may help PDIM transfer to host epithelial membranes is interesting. We agree that membrane mixing is most likely an important step for PDIM transfer, however, if mixing were the deterministic mechanism driving lipid transfer then we would expect trehalose monomycolate to also be able to spread into epithelial cells, which it does not. Additionally, our data demonstrating that methyl-branched mycocerosic acids promote PDIM fluidity and are required for spreading into epithelial membranes supports the hypothesis that the biophysical properties of PDIM on the bacterial surface promote its ability to spread into host membranes.dim’s Fluidity Promotes Spread into Epithelial Cell Membranes”.Regarding the different kinetics of spreading between epithelial cells and macrophages, we do not believe our data suggest that the rate of spreading is different between the two. In fact, our data cannot speak to the rate of lipid spread into host membranes. Our data shows that PDIM spreads into epithelial membranes before it spreads into macrophage membranes. We believe this is a result of the physiology of infection. In our infection model, mycobacteria remain in an extracellular space for around 3-5 hours following infection after which they are phagocytosed by macrophages. While in this extracellular space they can make contact with epithelial cells. Thus, PDIM spreads into epithelial membranes prior to macrophage membranes because bacteria make contact with epithelial cells prior to interacting with/being phagocytosed by macrophages. A better explanation of these kinetics are now in subsection “PFor Figure 7, the rationale for the cholesterol study is interesting given that cholesterol is coupled to biosynthesis of methyl-branched lipids such as PDIM  and the role of DIM in macrophage invasion required cholesterol .We thank the reviewers for highlighting these important studies. We agree that the Wilburn review highlights an important link between cholesterol metabolism and PDIM bio-synthesis. We have included this in our Discussion. For the introduction to Figure 7 we were motivated by studies that suggested a physical association between PDIM and cholesterol outside of macrophages, as this suggested to us that cholesterol may be promoting the spreading into epithelial cells that we were observing.dim Spreads into Epithelial Membranes” we discuss these results. However, in contrast to Astarie-Dequeker et al., we found no difference in the rate of phagocytosis of wildtype or PDIM-deficient M. marinum following infection of zebrafish larvae . Therefore, we decided not to further describe the mechanism from Astarie-Dequeker et al., where they show cholesterol being required for PDIM-promoted phagocytosis as we felt it may not explain the results in our model.The work from Astarie-Dequeker et al., does more directly demonstrate an interaction between PDIM and cholesterol. Indeed, insubsection “PFigure 7A-D: Can the distinct hydrophobicity between M. marinum WT and mmpL7 mutant influence their capacity to sequester cholesterol. The authors could consider testing another hydrophobic lipid to further underscore this point.M. marinum could sequester 16:0 azidocaproyl phosphoethanolamine . We found that either strain were capable of sequestering this hydrophobic compound , suggesting that there is not dramatic differences in the hydrophobicity of wildtype and ∆mmpL7 M. marinum.The reviewers raise an excellent point. The ability of mycobacteria to sequester cholesterol could be due to their hydrophobic outer cell membrane and not due to interactions with PDIM directly. To test this idea we chose a compound that was just as hydrophobic as cholesterol but structurally very distinct. We tested whether wildtype or ∆mmpL7 3) Crucial data is also unnecessarily buried in supplementary figures. Supplemental Figure 3 is important to the interpretation of the results and needs to be more visible as part of a main figure. As there is no display item limit, so consider integrating the "validation" data into Figure 3 or splitting up Figure 3. Finally, the authors should compare their current results more extensively to previous findings to better illustrate the broader context of the topics addressed.We appreciate that the reviewers consider our supplemental data important to the understanding of our manuscript. As such we have taken their advice and have added these panels to the main figures and have split figure three into two figures.The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:The reviewers appreciate the careful attention of the authors to address most of the original comments, which has provided stronger support of their interpretations and conclusion. However, there are a few points that still need attention as detailed here. These comments should be able to be addressed by changes in the text.1) Claims of TLR inhibition (starting in the abstract and throughout the manuscript) need to softened or removed as this is not directly addressed in the manuscript. The authors show effects on monocyte recruitment and that MyD88 deletion affects monocyte recruitment, but the experiments do not directly show PDIM is inhibiting TLRs and really just show that MyD88 is required for monocyte recruitment.The claims of TLR inhibition have been softened and instead are more focused on Myd88 signaling and the resulting recruitment of microbicidal monocytes. These changes were made throughout the manuscript.2) The authors should comment on the membrane effects of PDIM in the context of the literature . These recent data obtained with PDIM of M. tuberculosis would provide strong support for their findings.We have discussed these works in the Discussion.3) Introduction: Could the authors clarify what fluidity means for a lipid? Membrane fluidity and lipid mobility may be more appropriate.You are correct and this language has been corrected throughout the manuscript.4) Subsection “Chemical Extraction and Reconstitution of the Mycomembrane of M. marinumof the Mycomembrane of M. marinum”: The authors clearly showed that by mixing of extracted lipids with bacteria, the lipids coat M. marinum, but the authors do not demonstrate that the mycomembrane is reconstituted. For instance, what is the localization and orientation of lipids once coated on the bacteria in comparison to native lipids? This can be addressed by more specifically writing "coating of bacteria with extracted lipid" instead of "reconstitution of the mycomembrane".Reconstitution has been replaced with recoating throughout the manuscript.5) subsection “Pre-infection PDIM Reservoirs are Required for Virulence”: The authors should mention and discuss that deletion of mas or mmpL7 also results in defects in PGL synthesis and localization.We have brought this to the reader’s attention (subsection “PDIM Spreads into Macrophage Membranes”).6) Subsection “PDIM Spreads into 148 Host Macrophage Cell Membranes” and thereafter: The authors should define more clearly the notion of spreading. Did Azido-DIM spread within the same membrane, suggesting a lateral diffusion, and/or propagate to distinct membrane compartments?Our data suggests both to be taking place. We see spreading appearing next to where bacteria make contact with host membranes as well as intracellular punctate forming. We have made clearer these observations .7) subsection “PDIM’s Fluidity Promotes Spread into Epithelial Cell Membranes”: The authors claimed that PDIM's fluidity correlates with the lipid's ability to spread into host membrane. Another possibility could be that PDIM's ability to diffuse into the bacterial membrane increases the membrane fluidity, which facilitates membrane mixing. The consequence would be the transfer of PDIM to the host membrane, in which the lipids inserted and can diffuse laterally, as previously reported . The effect of PDIM on bacterial membrane fluidity could therefore be an explanation for the transfer of the lipid rather than for its spreading. In contrast, TMMs are less capable of diffusing and getting transferred. TMM are amphiphilic molecules in comparison to the highly hydrophobic PDIM. This strongly suggests a relationship between lipid structure and capacity to get transferred to host membranes. Please discuss this point in the Discussion section.We have expanded on how our data speaks to these more basic principles of lipid dynamics (Discussion).8) The authors should be clear in their conclusions and the Discussion that the data have been obtained with bacteria coated with extracted lipids.We make have made this clear in the Discussion.9) The additional images provided by the authors are encouraging but there is not sufficient detail about the quantification to understand if this is representative or not. Furthermore, while the authors are showing overlapping signals with the plasma membrane, these images do not prove that PDIM is in the membrane. To show that PDIM is in the membrane, a z stack is required but not provided.We have included a z projection of DIM-488 spreading into the plasma membrane of A549 cells. .


Pulse oximetery is expected to be an indirect estimation of arterial oxygen saturation. However, there often are gaps between SpO2 and SaO2. This study aims to study on arterial oxygen saturation measured by pulse oximetry and arterial blood gas among patients admitted in intensive care unit.It was a hospital-based descriptive cross-sectional study in which 101 patients meeting inclusion criteria were studied. SpO2 and SaO2 were measured simultaneously. Mean±SD of SpO2 and SaO2 with accuracy, sensitivity and specificity were measured.According to SpO2 values, out of 101 patients, 26 (25.7%) were hypoxemic and 75 (74.25%) were non-hypoxemic. The mean±SD of SaO2 and SpO2 were 93.22±7.84% and 92.85±6.33% respectively. In 21 patients with SpO2<90%, the mean±SD SaO2 and SpO2 were 91.63±4.92 and 87.42±2.29 respectively. In 5 patients with SpO2 < 80%, the mean ± SD of SaO2 and SpO2 were: 63.40 ± 3.43 and 71.80±4.28, respectively. In non-hypoxemic group based on SpO2 values, the mean±SD of SpO2 and SaO2 were 95.773±2.19% and 95.654±3.01%, respectively. The agreement rate of SpO2 and SaO2 was 83.2%, and sensitivity and specificity of PO were 84.6% and 83%, respectively.Pulse Oximetry has high accuracy in estimating oxygen saturation with sp02>90% and can be used instead of arterial blood gas. As PO is a non-invasive device, it can be used instead of ABG. Some studies suggest that this method does not exactly reflect the values of ABGs.7.Pulse oximetry (PO) is a useful tool for clinical and investigational purposes for indirect measurements of oxygen saturation.12, because as SaO2 decreases, bias will be increased, while precision (the standard deviation of the differences) will be decreased, with SpO2 increasingly overestimating SaO2.13The majority of patients admitted in ICU show gaps between arterial oxygen saturation measured by ABG and PO. Many studies suggest that pulse oximeters are inaccurate at low saturationsThis study aims to study on arterial oxygen saturation measured by pulse oximetry and arterial blood gas among patients admitted in intensive care unit.A descriptive cross-sectional study was conducted in ICU of Bir Hospital, Mahaboudha, Kathmandu from Oct 2017 to Oct 2018 after taking ethical clearance from Institutional Review Board, National Academy of Medical Sciences (Ref: 1054). History, clinical examination, chest X-ray findings, pulse oximetry and ABG were reviewed. Patients admitted in ICU with or without hypoxemia during the study period were included in the study after obtaining informed consent.Inclusion CriteriaPatient older than 18 years admitted in ICU.Arterial blood gas analysis and pulse oximetry should be taken simultaneouslyInformed consent is given by the patient (and from the relatives of the patients who were unconscious).Exclusion criteriaData were excluded when SaO2 was less than 60% or PaO2 were higher than 100mmHg.Patients under 18 years of age.Venous blood sampleConvenient sampling was done and the sample size was calculated using the formula,Z = 1.96 ( reliability coefficient at 95 % confidence level)p = 94 (prevelance of study)q = 100-pd = maximum tolerable error = 5Z = 1.96 at 95 % CIwhere,The calculated minimum sample size was 86.6, however, the total sample size taken was 101. Data were collected using a structured proforma covering the relevant details.ABG and pulse oximetry were taken simultaneously in all ICU admitted patients Arterial blood sample (about 0. 5ml) was obtained from the radial artery following confirmation of collateral vessel flow by Allen's test or modified Allen test. These are bedside tests that demonstrate collateral flow through the superficial palmar arch.Before taking the sample, the syringe lumen was heparinized (0.1 ccs). Air bubbles, if present, was immediately expelled from the sample; the sample was sealed in an iced container and taken to blood gas analyzer .SpO2 was obtained using a pulse oximeter . The finger probe for the unit was placed on the index finger of the opposite arm from which the arterial sample had been taken. Spo2 was measured twice at 0 minutes and 2 minutes, then the average of two was taken. In this study, ABG values were taken as reference values. According to SpO2, the studied patients were divided into three groups: SpO2 <80%, 90%>Sp02≥80% and SpO2 ≥ 90%. Hypoxemia was considered as SpO2 or SaO2 <90% or partial pressure of oxygen (Pa02) < 60mm Hg by ABG.The data were collected and entered in MS-Excel 2007 and analyzed using the IBM Statistical Package for Social Sciences (SPSS) version 20 software. Sensitivity, specificity and accuracy of PO were measured and calculated.A total of 101 patients were enrolled in this study . Twenty six (25.7%) were hypoxemic and 75 (74.25%) were non-hypoxemic according to PO and based on ABG results, SaO2 values were less than 90% in 13 (12.9%) patients, and the values were equal or more than 90% in 88 (87.1%) patients. The mean ± SD oxygen saturation values (SaO2) measured by ABG analyzer system were greater than those measured by pulse oximetry (SpO2): .There were differences between pulse oximetry (SpO2) and ABG values (SaO2) in the limit of SpO2<80% and 90% >SpO2 ≥80% .In 21 hypoxemic patients (SpO2<90%), the mean ± SD SaO2 and SpO2 were: 91.63±4.92 and 87.42±2.29. In 5 of the hypoxemic patients (SpO2 < 80%), the mean ± SD of SaO2 and SpO2 were: 63.40±3.43 and 71.80±4.28, respectively. As patients were defined non-hypoxemic based on SpO2 values, the mean ± SD of SpO2 and SaO2 were 95.773±2.19%, 95.654±3.01%, respectively. The sensitivity and specificity of pulse oximetry were 84.6 % and 83 %, respectively .In acute illness, patients with SpO2 ≥ 90%, PO has high accuracy in estimating SaO2 and may be used instead of ABG. The study suggests that in patients with SpO2 < 90%, however, the exact estimation of SaO2 and the evaluation of oxygenation by pulse oximeter is not a good substitution for ABG analyzer.Regarding the comparison between oxygen saturation measured by pulse oximetry and ABG, many studies have been conducted related to accuracy.14 In the study of Carter et al., the performance of pulse oximetry deteriorated below a SpO2 of 75%.15 In the study of Chiappini et al., a significant difference was found between SpO2 and SaO2 values.16 SpO2 values were lower than SaO2 (90.58±5.45% vs. 92.14±5.79%) and a lack of accuracy of the pulse oximeter was found, but only for SpO2 values below 82%.In a recent meta-analysis of the measurement of SaO2 by pulse oximetry, Jensen et al. concluded that, from the 74 studies (1976 to 1994 ), pulse oximeters were accurate within 2% in the range of 70-100% SaO2.17Study of Kenneth P Levin et al conducted in 2001, shows that increasing the assessment of arterial oxygenation among patients with community-acquired pneumonia(CAP) is likely to increase the detection of arterial hypoxemia, particularly among outpatients. However, ABG should be considered for patients with underlying cardiopulmonary disease, for those for whom ventilatory failure is of clinical concern, and if other reported sources of error for PO are present.18 The accuracy of pulse oximetry was 90.8%, sensitivity was 93.3% and specificity was 89.1%. The study reported that in pulmonary diseases with SpO2 ≥ 80%, PO has high accuracy in estimating SaO2, which is contradictory to our study where accuracy is estimated when Spo2 > 90%. In patients with SpO2 < 80%, however, the result is similar to our study and concluded that the evaluation of oxygenation by pulse oximeter is not a good substitution for ABG analyzer.Similarly, another study of Ebrahim Razi, Hossein Akbari in 2006 done for COPD patients included 152 patients.19In a study of Kanai R., Moriyama K. et al done in large scale including 20717 arterial blood gas samples from ICU patients, SpO2 tended to show a higher value than SaO2 and suggest to keep SpO2 above 92% to avoid hypoxemia in the ICU to decrease morbidity and mortality.20 In the study of Webb et al., nearly 30% of values reviewed were erroneous by more than 5% at saturation of less than 80%.Webb et al. reported that pulse oximetry is poorly calibrated at low saturations and generally less accurate and less precise than at normal saturations.12, because as SaO2 decreases, bias will be increased, while precision (the standard deviation of the differences) will be decreased, with SpO2 increasingly overestimating SaO2.13 Many explanations have been proposed for the limited performance of pulse oximeters at low saturations. One is the slight variations in the output wavelength of the light-emitting diodes which generate proportionally larger errors at low saturations.22 Another is the generation of proportionally larger errors in the measurement of transmitted red light versus of infra-red light at low saturations because of the large extinction coefficient of reduced haemoglobin.23Many studies suggest that pulse oximeters are inaccurate at low saturationsIn the current study when oxygenation saturation was more than 90%, there was good correlation coefficient between the two methods of measurements . The study revealed that only two patients (15.4%) out of all who were considered non-hypoxemic according to PO were considered hypoxemic in terms of ABG values. The obtained agreement rate was 83.2%. Given the critical point of Sp02≥90%, about 83.2% of cases correlated this regard. Thus, although patients were considered as hypoxemic or non-hypoxemic according to pulse oximetry or ABG results, because of slight changes among the results of the two methods and also a wide variety of cases, correlation coefficient had shown good correlation between two methods, especially in Spo2≥90% with P<0.001.From the above discussion, we can conclude that PO is easily available, painless, non-invasive continuous monitoring and cheap method that can be considered an appropriate substitute for ABG, especially in SpO2 ≥90% with an accuracy of about 83.2%. However in conditions with low oxygen saturation (SpO2 <90%) and in critical status, PO is a poor predictor of hypoxemia or not a good alternative to estimate arterial oxygen saturation. There are several limitations of the study as the sample size is small and the accuracy rate is only 83.2%. Futher studies are required for accuracy and effectiveness of the PO in a large sample. In addition, though PO measures arterial oxygenation, it does not assess ventilation. Therefore, it needs to be aided with ABG analysis when alveolar hypoventilation is suspected clinically.Pulse Oximetry has high accuracy in estimating oxygen saturation with sp02>90% and can be used instead of arterial blood gas. The study suggests that in patients with SpO2 <90%, however, the exact estimation of SaO2 and the evaluation of hypoxemia by pulse oximeter is not a good alternative for ABG analyzer.


The objective of our study was to evaluate the association between perinatal asphyxia and hypoxic-ischemic encephalopathy (HIE) with the presence of ante and intrapartum risk factors and/or abnormal fetal heart rate (FHR) findings, in order to improve maternal and neonatal management.We did a prospective observational cohort study from a network of four hospitals  between 2014 and 2016. Neonates of gestational age ≥ 35 weeks, birthweight ≥1800 g, without lethal malformations were included if diagnosed with perinatal asphyxia, defined as pH ≤7.0 or Base Excess (BE) ≤ − 12 mMol/L in Umbical Artery (UA) or within 1 h, 10 min Apgar < 5, or need for resuscitation > 10 min.FHR monitoring was classified in three categories according to the American College of Obstetricians and Gynecologists (ACOG). Pregnancies were divided into four classes: 1) low risk; 2) antepartum risk; 3) intrapartum risk; 4) and both ante and intrapartum risk.In the first six hours of life asphyxiated neonates were evaluated using the Thomson score (TS): if TS ≥ 5 neonates were transferred to Hub for further assessment; if TS ≥ 7 hypothermia was indicated.p < 0.001, OR 0.59, 95% CI 0.45–0.79). 32/213 (15%) neonates were transferred from Spokes to Hub. Overall, 12/281 were treated with hypothermia. HIE occurred in 16/281 (5.7%) neonates: four grade I, eight grade II and four grade III. Incidence of HIE was not different between Hub and Spokes.Perinatal asphyxia occurred in 21.5‰ cases  and HIE in 1.1‰ . The total study population was composed of 281 asphyxiated neonates: 68/5152 (1.3%) born at Hub and 213/9744 (2.2%) at Spokes (Pregnancies resulting in asphyxiated neonates were classified as class 1) 1.1%, 2) 52.3%, 3) 3.2%, and 4) 43.4%. Sentinel events occurred in 23.5% of the cases and FHR was category II or III in 50.5% of the cases. 40.2% cases of asphyxia and 18.8% cases of HIE were not preceded by sentinel events or abnormal FHR.We identified at least one risk factor associated with all cases of HIE and with most cases of perinatal asphyxia. In absence of risk factors, the probability of developing perinatal asphyxia resulted extremely low. FHR monitoring alone is not a reliable tool for detecting the probability of eventual asphyxia. Cases of neonatal encephalopathy related to perinatal asphyxia are called hypoxic-ischemic encephalopathy (HIE) and can lead to long-term neurological sequelae and cerebral palsy , 2.The Sarnat and Sarnat classification  is stillBoth clinical  and insPerinatal asphyxia is defined as at least one of the following characteristics in a neonate: 10 min Apgar score ≤ 5, need for resuscitation > 10 min, metabolic acidosis  or within 1 h of life).There is a dose-dependent relationship between the degree of acidosis and the likelihood of adverse neonatal outcome , 6.Both ante and intrapartum risk factors have been associated with perinatal asphyxia and HIE. Some of these are clinically recognized as asphyxial birth events (sentinel events): uterine rupture, placental abruptio, shoulder dystocia and cord prolapse. However, the majority of risk factors  seem to interact in the causal pathway to HIE rather than being its direct antecedent –11. In aFetal heart rate (FHR) monitoring provides important information about fetal status. However, its role in the prediction of fetal asphyxia remains controversial, despite efforts directed at its interpretation and the development of protocols for the management of abnormal patterns , 15.To summarize, there are a number of conditions that have been associated with the presence of perinatal asphyxia and its extreme consequence, HIE. Nevertheless, it is problematic to identify the sequence of events that leads to perinatal asphyxia during prenatal and intrapartum care.In this context the objective of our study was to evaluate the association between perinatal asphyxia and HIE with the presence of risk factors and/or abnormal FHR findings in order to improve maternal and neonatal management.We conducted a prospective observational cohort study between July 2014 and June 2016 from a network of four hospitals with different levels of care which shared protocols of intrapartum and neonatal care.Inclusion criteria were asphyxiated neonates of gestational age ≥ 35 weeks, birthweight ≥1800 g, and no severe malformations. Perinatal asphyxia was defined as pH ≤7.0 or BE ≤-12 mMol/L in UA or within 1 h, 10 min Apgar ≤5, or need for resuscitation > 10 min .The network includes one Hub University Hospital center with a neonatal intensive care unit that offers therapeutic hypothermia, and three level I Community Hospital Spoke centers.In Spoke centers, only women with low risk pregnancies or minor complications can be admitted for delivery. High risk pregnancies for medical or fetal complications are referred to Hub. Patients with low risk pregnancies may choose to deliver in either Spoke or Hub centers.Perinatal mortality rate in our network is superimposable to the general Italian one, that is 4 ‰.The Italian childbirth system offers universal and free of charge maternity care.The acid-base status is routinely determined at birth by UA and, if not feasible, within one hour of life on the neonate.Data regarding maternal history, pregnancy complications, labor and delivery were collected.The uterine contraction changes during labor were monitored and prospectively registered. Tachysystole is defined as ≥5 contractions in 10 min for 30 min or more.Fetal electronic monitoring on admission and/or during labor was prospectively collected and managed using the Clark algorithm . The FHRWe divided pregnancies into four classes: low risk (no risk factors); antepartum risk ; intrapartum risk ; and both ante and intrapartum risk.Data recorded on each infant included: pH, BE, lactate, need for ventilation by mask or endotracheal tube during initial stabilization, need for cardiac massage, biochemical and instrumental data. In the first six hours of life, asphyxiated neonates were monitored with the Thomson score : if TS wEncephalopathy was classified according to Sarnat & Sarnat .Neurodevelopmental follow-up was offered to all neonates until 1 year of age and to neonates with moderate or severe encephalopathy until 6 years of age.Written informed consent was obtained from all participants.p value <.05 was considered significant. SPSS (IBM Corp. version 24) was used to run the analyses.Descriptive statistics were calculated as mean (standard deviation) for numerical variables and absolute frequencies and percentages for categorical variables. Statistical analysis included univariate analyses with Student T-test, one-way ANOVA, chi-square and Fisher exact test. Multivariate logistic regression analyses were performed to control for confounding variables (SPSS). A two-tailed During the study period perinatal asphyxia occurred in 321/14,896 cases (21.5‰) and HIE in 16/14,896 (1.1 ‰).Asphyxia occurred in 1.5% (79/5152) in the Hub center, in 1.8% (64/3552) in Spoke 1, 2.9% (94/3269) in Spoke 2 and 2.9% in Spoke 3 (84/2923).p < 0.001, OR 0.59, 95% CI 0.45–0.79).We excluded 40 neonates because they did not meet the inclusion criteria , or because we did not have parental consent, or we had incomplete data for enrolment. Thus, 281 asphyxiated neonates were included in the analysis: 68/5152 (1.3%) delivered at the Hub and 213/9744 (2.2%) in Spoke centers , 261/281 had BE ≤ − 12 (93%) (of which 51 (18%) with BE ≤ − 16); 189/281 (67%) neonates were enrolled only for BE ≤ − 12.There were no cases of HIE among neonates who were not enrolled.There were no perinatal deaths during the study period.Descriptive characteristics of asphyxiated neonates are reported in Table During neonatal monitoring TS was ≥5 in 24/281 cases (8.5%) and 32/213 (15%) neonates were transferred from Spokes to the Hub Center.p = 0.58, OR = 1.4 95% CI 0.44–49. The detail of every case is reported in Table Overall, 12/281 neonates received hypothermia. There were 9/281 cases of neonatal seizures. HIE was diagnosed in 16/281  including four with grade I, eight with grade II, and four with grade III HIE. Asphyxia and HIE according to place of birth are shown in Fig. Pregnancies were classified as low risk in 3/281 (1.1%) cases, with antepartum risk in 147/281 (52.3%), intrapartum risk in 9/281 (3.2%), and both ante and intrapartum risk in 122/281 (43.4%) cases  nulliparous women equally distributed in class 2 and 4, 16/281 (5.7%) women > 40 years, 87/281 (31.0%) with BMI > 30, 14/281 (5.0%) cases of oligohydramnios, 20/281 (7.1%) cases of polyhydramnios and 8/281 (2.8%) cases of fetal growth restriction. We recorded 18/281 (6.4%) cases of hypertensive disorders of pregnancy, 4/281 (1.4%) positive thrombophilic status, 5/281 (1.8%) pre-gestational and 31/281 (11.0%) gestational diabetes mellitus, 25/281 (8.9%) thyroid diseases, 8/281 (2.8%) gestational cholestasis, 1/281 (0.4%) antepartum hemorrhage and 19/281 (6.8%) previous cesarean section.Labor was induced in 96/281 (34.2%) of cases. Thick meconium during labor was observed in 21/281 cases (7.5%), fever or chorioamnionitis in 17/281 (6.0%) and tachysystole in 52/281 (18.5%).p = 0.4089).Sentinel events occurred in 66/281 cases 23.5%) and preceded HIE in 6/16 (37.5%), as described in detail in Fig. .5% and pOnly three cases of asphyxia (1%) and no cases of HIE occurred in class 1 (low risk pregnancies). There were no cases of UA pH ≤ 7 in this class.There were 67/281 (24%) operative vaginal deliveries and 56/281 (20%) cesarean sections including 39/56 (70%) because of fetal distress. FHR category II and III in the last hour before delivery were documented in 142/281 (50.5%) cases of perinatal asphyxia, and they preceded HIE in 9/16 (56.3%) respectively. In 7/142 cases, FHR category II or III was observed before labor.p < 0.001 OR 4.8 95% CI 2.6–8.9). HIE occurred more often if UA pH was ≤7  in respect to case with UA pH > 7  .Moreover, FHR category II or III occurred in 61/80 (76%) cases with UA pH ≤ 7 compared with 81/201 (40%) cases with UA pH > 7 (113/281 (40.2%) cases of asphyxia and 3/16 (18.8%) cases of HIE were not preceded by sentinel events or abnormal FHR findings.In our network the rates of neonatal asphyxia (1%), as well as of pH ≤ 7 5‰) and HIE (0.7–1.7‰) were comparable to those described in literature when similar definitions are used ‰ and HIE, 18–22.As expected, only a minority of asphyxiated neonates developed HIE (5.7%) and they were promptly identified. Thanks to our shared care protocols and cooperative management system, appropriate care was guaranteed for all asphyxiated neonates, while minimizing the number of newborns transferred.In our series the number of sentinel events in cases of perinatal asphyxia and HIE was as high as previously described in literature (15–35%) , 13. EmeMany authors have focused on searching for risk factors associated with perinatal asphyxia and neonatal encephalopathy: a wide number of antecedents has been identified, nonetheless a variable percentage of the events remain unexplained , 9, 11. Our study design does not allow us to assign a relative risk or hazard ratio to the risk factors recorded.The most common risk factor for asphyxia is nulliparity. It is identified in 76% of cases  and it is the only risk factor in 16% of cases. This is obviously a non-modifiable condition and it is widespread in the general obstetric population. Nonetheless, it has been previously described in association with need for cooling , 22. TheIn spite of this, a recent study of Liljestrom  reportedThe second most frequent risk factor is fetal tachysystole, which presented in 52/281 cases (18.5%). In most cases, tachysystole was associated with other risk factors, such as the need for induction of labor (23 cases) and/or oxytocin augmentation (29 cases). Use of oxytocin can lead to excessive uterine activity, which has been associated with neonatal morbidity both in spontaneous and induced labor . Unlike From the review of FHR tracing prospectively collected and managed according to ACOG algorithms, we observed that FHR Category II or III occurred in 50.5% of asphyxiated neonates. It preceded HIE in 56.3% of cases.As recently reported , we acknIndependently from the efforts at training and at applying uniform algorithms during labor, it may be unrealistic to expect high sensitivities for the detection of asphyxia using FHR alone. This underscores thefact that key management decisions in preventing asphyxia should not be based only on FHR interpretation. Other factors, including maternal and fetal history, stage and progress of labor and signs of fetal distress in labor, should be taken into account. Some Authors have suggested that a physiological interpretation of the FHR recordings may improve our understanding offetal oxygenation during labor , 27. WheOur study has some limitations. First of all, the number of cases included in the study is not large. In addition, some cases were excluded due to lack of information or refusal of consent. The strengths of our study are that umbilical cord gas analysis was done consistently across the participating institutions; and the prospective nature of the study, with hospitals using shared protocols of maternal and neonatal care, as well as of methods of induction, labor management, oxytocin use, and FHR tracing interpretation.Furthermore, all FHR tracings were reviewed in order to standardize their interpretations. In addition, for every neonate we have the values of both pH and BE and lactate, thus permitting to have a more realistic picture of all nuances of acid-base status, and not only the most extreme. In our network, all cases of HIE were promptly identified with a limited number of newborns transferred. Shared care protocols and cooperative management system has guaranteed appropriate care for all asphyxiated neonates. We hope that long-term neurodevelopmental follow up data will help us to better define risk factors for neurological sequelae in asphyxiated neonates, allowing the development of adequate rehabilitation programs for these children.We identified at least one risk factor associated with all cases of HIE and with the majority of cases of perinatal asphyxia. In the absence of risk factors, the probability of developing perinatal asphyxia resulted extremely low. Among risk factors, the most frequent were nulliparity and tachysystole. Sentinel events confirmed their significant role as antecedents of asphyxia. Our findings on FHR tracing support that it is unrealistic to expect high sensitivities for the detection of asphyxia using FHR alone. FHR interpretation should consider the background risk factors. Future studies will address the question of whether a more integrated approach taking into account risk factors and new adjunctive monitoring methods can improve awareness of fetal status and, consequently, prediction of asphyxia.


The level of statistical significance was set at p < 0.05. Results: A total of 1808 patients with GD with a median age of 44 (range 14–85) years were enrolled in a 25-month period by 78 departments, of which 35.7% (n = 645) had an endocrine orbitopathy and 0.1% (n = 6) had thyrotoxic crisis. Conventional open surgery was used in 98.6% of cases and minimally invasive or remote-access approaches were used in 1.4%. Total thyroidectomy was performed in 93.4% of cases (n = 1688). Intraoperative neuromonitoring (IONM) was used in 98.9% (n = 1789) of procedures. In 98.3% (n = 1777) at least one parathyroid gland was visualized and in 20.7% (n = 375) parathyroids were autografted. The rates of unilateral and bilateral transient RLNP were 3.9% (n = 134/3429 nerves at risk) and 0.1% (n = 4/3429 NAR). The rates of transient RLNP tended to be higher when intermittent IONM was used compared to continuous IONM . The rate of transient postoperative hypoparathyroidism was overall 29% (n = 525/1808). Multivariate analysis revealed fewer than 300 thyroid resections and fewer than 15 thyroid resections for GD per year, male sex, BMI > 30, autotransplantation of parathyroid glands and previous bilateral thyroid surgery as independent risk factors for postoperative temporary hypoparathyroidism. Reoperations for bleeding (1.3%) were rare. Conclusion: Total thyroidectomy with IONM is safe and currently the most common surgical therapy for GD in Germany. Postoperative hypoparathyroidism is the major complication which should be focused on.Background: Surgical treatment of Graves’ disease (GD) has a potentially increased incidence of postoperative hypoparathyroidism, recurrent laryngeal nerve palsy (RLNP) and bleeding. The aim of this study was to evaluate the current extent of surgery for the treatment of GD and its safety as a short-term outcome. Methods: Patients who underwent thyroid resection for GD were identified from the prospective StuDoQ/Thyroid registry. Patient data were retrospectively analyzed regarding demographics, surgical procedures and perioperative outcomes. Statistics were performed with Student’s  Graves’ disease (GD) is an autoimmune disorder caused by the production of auto-thyrotropin receptor antibodies against the thyroid-stimulating hormone (TSH) receptor . GD is tThe major complication of thyroid resection for GD is postoperative hypoparathyroidism with transient and permanent rates between 18.0–20.0% and 3.0–7.0% ,15,16,17The aim of this study was to evaluate the current surgical strategy for the treatment of GD in Germany and its short-term outcomes.www.dgav.de), and the EUROCRINE registry (https://eurocrine.eu). A total of 52 parameters were defined by a browser-based electronic case report form (eCRF). Data from participating centers were entered in pseudonymized form at the institutional level without an over-institutional identifier in the prospective StuDoQ/Thyroid registry. The data were immediately subjected to an automatic plausibility check with automatic error indication.The StuDoQ/Thyroid Registry of the German Society of General and Visceral Surgery (DGAV) was designed as a prospective, multicenter database with web-based data entry, has been maintained since April 2017 and was developed in close cooperation with the board of the German Society of Endocrine Surgeons (CAEK). Relevant outcome and quality indicators were selected on the basis of the German S2k guidelines ,20 for tAll participating institutions had to approve the correct number of thyroid resections entered in the registry by a written confirmation of the hospital’s administration. In addition, the completeness and correctness of the data was checked as part of the regular certification audits for the certified competence and reference thyroid centers of the DGAV, and by external monitoring since 2019.All enrolled patients signed an informed consent for data collection and approval for data safety management was obtained from the TMF (Technologie und Methodenplattform für die vernetzte medizinische Forschung).For the present study, all patients who underwent thyroid resections for Graves’ disease between 1 April 2017 and 30 April 2019 were retrieved from the StuDoQ/Thyroid registry. The diagnosis Graves’ disease was preoperatively based on clinical symptoms of hyperthyroidism, an existing EO, laboratory thyroid function tests as thyroid stimulating hormone (TSH), free triiodothyronine (fT3), thyroxine (fT4) and TSH receptor antibodies, with respect to concomitant substitution or thyrostatic medications to achieve euthyroidism. Patients were considered hyperthyroid with preoperative TSH levels <0.1 mU/L or low TSH levels (0.02–0.34 mU/L). GD was postoperatively confirmed by the pathological analysis. The indications for operation as definitive treatment of GD were according to the national S2k-guideline “operative treatment of benign thyroid diseases”  thyroid The postoperative function of the recurrent laryngeal nerve was determined by laryngoscopy within the first 5 postoperative days by an independent surgeon or head and neck specialist as required in the national S2k-guideline “operative treatment of benign thyroid diseases” . Vocal ct-test and qualitative variables  using the chi-square test or Fisher’s exact test as appropriate. The effect of single risk factors on hypoparathyroidism was explored in bivariate logistic regressions. A multivariate logistic regression model was analyzed using stepwise regression.Continuous variables are presented as means (standard deviation) and categorical variables as proportions. Quantitative variables were compared using the Student’s p-values) are based on two sided tests; the level of statistical significance was set at p < 0.05. Analyses were performed using SPSS 23.0 .All reported probability values (n = 1429) of patients with GD were operated in hospitals with more than 300 thyroid resections per year; only 4.9% (n = 88) of patients were operated on in hospitals with 100 or fewer thyroid resections per year. A total of 11 institutions enrolled more than 50 patients, 7 institutions 31–50 patients and 9 institutions 21–30 patients with GD, whereas the remaining 51 institutions operated on 20 or fewer patients with GD during the recruitment period  patients had Graves’ disease (GD), who were recruited by 78 (87%) surgical departments. The data of these patients, who were all discharged until April 30th, 2019, were analyzed. A total of 79% (t period .n = 1478) of women and 18.3% (n = 330) of men with a median age of 44 (range 14–85) years underwent thyroid resection for GD. Only 2.4% (n = 43) of patients were younger than 18 years and 3.5% (n = 64) patients were older than 70 years. Furthermore, 7.2% (n =131) of patients  were overweight with a body mass index > 35 (n = 193) of patients had an increased perioperative risk with an ASA (American Society of Anesthesiologists) score ≥ 3. A total of 35.7% (n = 645) of patients had endocrine orbitopathy and 55.3% (n = 999) of patients had biochemical hyperthyroidism (TSH < 0.01 mU/L or low TSH 0.02–0.34 mU/L) at the time of surgery. A total of 1536 of 1808 patients (84.9%) took preoperative antithyroid drugs. Furthermore, 99.7% (n = 1802) of patients underwent elective surgery and 0.3% (n = 6) had emergency surgery for thyrotoxic crisis. A total of 96.2% (n = 1740) of patients had initial surgery and 3.7% (n = 66) patients had cervical reoperations. Another two patients (0.1%) underwent radiofrequency ablation of GD prior to surgery. Preoperative laryngoscopy for the assessment of vocal cord function was performed in 98.5% (n = 1780) patients. Normal results were obtained in 98.1% (n = 1774) of patients and 0.3% (n = 6) of patients showed a unilateral palsy of the recurrent laryngeal nerve (RLN). Patient characteristics are summarized in A total of 81.7% , whereas bilateral subtotal resections  and Hartley-Dunhill procedures  were an exception (n = 98) of the procedures were hemithyroidectomies, mainly as reoperations for either previous neck surgery or as incomplete resections because of suspected RLN damage after resection of the first side (n = 1782) of patients, conventional open surgery was performed, whereas 1.3% (n = 24) had a minimally invasive video-assisted thyroidectomy (MIVAT), and 0.1% (n = 2) of patients had a robotic-assisted transaxillary thyroidectomy. Other endoscopic or robotic-assisted remote-access approaches like BABA  were not used. Only 0.05% (n = 1) of patients required a partial sternotomy for the resection of GD. In 5.4% (n = 97) patients any type of selective or systematic lymphadenectomy was performed, which showed no influence on postoperative hypoparathyroidism (p = 0.63) or recurrent laryngeal nerve palsy (p = 0.58).The vast majority of patients underwent total thyroidectomy  of patients. Intraoperative neuromonitoring (IONM) of the RLN was used in 98.9% (n = 1789), and intermittent IONM  was significantly more often used than continuous IONM . The rate of transient RLN palsy (per NAR) after use of continuous IONM was 3.4% compared to 4.1% when intermittent IONM was used  of patients.Visualization of the recurrent laryngeal nerve (RLN) was performed in 99.8% (n = 1777) of patients at least one parathyroid gland was identified intraoperatively (n = 1747) two or more parathyroids were visualized. Autotransplantation of parathyroid glands was undertaken in 20.7% (n = 375) of patients. In the vast majority of these patients , two parathyroid glands were autotransplanted.In 98.3%  weighed less than 10 g and 4.9% (n = 88) more than 100 g. Histopathological examination confirmed GD in all patients; one (0.05%) patient showed, in addition to GD, a papillary carcinoma.The median specimen weight was 24 (range 8–242) grams, whereby 4.6% (n = 134 NAR) and bilateral dysfunction in 0.1%  NAR, and 40% (n = 40) of these nerves showed a permanent unilateral palsy, whereas no bilateral dysfunction was present. Based on a follow-up rate of 80%, one can extrapolate a rate of permanent RLNP of about 1.5% per NAR (50/3332).Postoperative laryngoscopy was performed in 97.2% (3332 of 3429 NAR) of nerves at risk (NAR). This revealed a vocal cord dysfunction in 4.0% (138 of 3332 NAR), with unilateral dysfunction in 3.9% (n = 525/1808) of patients; in 2.3% (n = 42) of patients even intravenous calcium substitution was supplemented. A total of 354 (67.4%) patients with postoperative hypoparathyroidism (n = 525) received simultaneous vitamin D at discharge. The rate of early postoperative hypoparathyroidism was independent of the number of parathyroid glands visualized intraoperatively (p < 0.0001). The rate was highest after autotransplantation of more than two parathyroid glands , center volume of more than 30 thyroid resections for GD during the study period (p < 0.0005), sex (p < 0.0005), age (p < 0.0005), BMI (p = 0.019), autotransplantation of parathyroid glands (p < 0.0005) and previous bilateral thyroid surgery (p = 0.028) showed a statistically significant influence on postoperative hypoparathyroidism in the univariate logistic regression, which was confirmed in the multivariate Cox regression analysis  of patients. These included postoperative bleeding in 1.3% (n = 23), wound infection in 0.2% (n = 3) and other reasons such as lymphatic fistula in 0.2% (n = 3), respectively. Another 14 (0.8%) patients underwent complete of thyroidectomy 2–4 days after the initial operation, since the first procedure was terminated as hemithyroidectomy due to suspicion of RLNP by IONM, which was not confirmed during postoperative laryngoscopy.Reoperations for complications had to be performed in 1.6%  or remoIn the present study, total thyroidectomy was by far the most frequently performed procedure 93.4%) to treat GD, whereas bilateral subtotal resections 0.6%) and Hartley-Dunhill procedures (0.8%) were rarely used. This trend has already been observed in an earlier European questionnaire study covering the years 2010 to 2012 in 2488 patients with GD . This st.4% to tr% and HarTotal thyroidectomy is regarded as an effective treatment of GD with definitive control of hyperthyroidism and its symptoms ,29. Subtn = 327). In the present study, parathyroid autotransplantation was associated with higher risk of postoperative transient hypoparathyroidism , which was highest after autotransplantation of two or more parathyroid glands. Former retrospective analyses of thyroid surgery confirmed parathyroid autotransplantation as an independent risk factor for postoperative transient hypoparathyroidism in patients undergoing thyroid surgery for autoimmune thyroid disease [Transient postoperative hypoparathyroidism is caused by transient reduced vascularization or even accidental removal of parathyroid glands. Thus, guidelines recommend the identification and protection of at least one parathyroid gland during surgery for GD . In the  disease ,36,37,38 disease . Another disease ,40,41,42One serious complication after thyroid surgery for GD is probably permanent postoperative hypoparathyroidism. In the present study, permanent postoperative hypoparathyroidism was extrapolated in approximately 2.9% of patients. A European questionnaire study of 1141 patients with GD reported a rate of permanent hypoparathyroidism of 2.8% . The mosPatient-specific independent risk factors for postoperative temporary hypoparathyroidism were female gender, age < 50 years, BMI > 30 and previous bilateral thyroid or parathyroid surgery; former data gave similar results ,38,48,49p = 0.058) [p = 0.059). Thus, the present StuDoQ data support the hypothesis that continuous IONM might be superior to intermittent IONM to prevent transient RLN palsy in GD. This hypothesis has to evaluated in the planned multicenter randomized controlled CITY trial.Although IONM is not recommended by the German  nor othe= 0.058) . The preThe rate of reoperations due to postoperative bleeding after thyroid surgery for GD was 1.3%, which is in the range present in the literature (0.5 to 6.8%) ,14,32.A recent systematic review involving 10,743 patients with GD and 3336 non-GD patients with hyperthyroidism showed that the pooled prevalence of incidental thyroid carcinoma was 7.0% , which iThe present study has some drawbacks and some strengths. The analysis has the limitation of being a retrospective study, although the data are prospectively documented, externally monitored by independent surgeons and the respective departments that control of the participating centers. The 6-month follow-up data regarding RLNP and postoperative hypoparathyroidism were not available for about half of the patients, so that at present no definitive numbers can be given for permanent RLNP and postoperative hypoparathyroidism. However, the present study based on the StuDoQ/Thyroid registry gives a clear picture of the actual trends of thyroid surgery for GD in Germany, since about 20% of operated patients with GD in Germany were documented in 25 months.Total thyroidectomy with IONM is the current most common surgical therapy for GD in Germany. The procedure is safe; however, postoperative hypoparathyroidism is the major complication, thus requiring more awareness.