World Champion outsprints Vos for her first career stage win at Italian stage race In almost an exact repeat of the 2021 World Championships sprint Balsamo went head-to-head in a sprint with cyclo-cross World Champion Marianne Vos (Jumbo-Visma) on the finishing straight in Tortolí but the Italian edged the Dutch rider on the line.  Balsamo’s teammate Elisa Longo Borghini – who recently showed her own sprinting prowess at The Women’s Tour – acted as last woman for the younger rider bringing her around the left hand side in order to get a clear run at the line.  Vos launched her sprint first but it wasn’t long before the world champion was on her wheel choosing the right moment to come around the Jumbo-Visma rider and throw her bike to the line to claim the victory.  Charlotte Kool (Team DSM) came from a position much further back to claim third on the stage.  The win sees Balsamo take the pink leader’s jersey from Team BikeExchange’s Kristen Faulkner Balsamo leads by four seconds ahead of Faulkner "Wearing the maglia rosa is a dream come true wearing it tomorrow will be beautiful," Balsamo said.  "We came here with the aim of not taking too many seconds in the prologue and then try to take the jersey with bonuses we took responsibility from the first to the last km Tomorrow we will try to defend the [jersey] but we have no particular pressure On the first road stage of the 2022 Giro d’Italia Donne the peloton tackled a 106.4km rolling point-to-point stage from Villasimius to Tortolì just inland from Sardinia’s eastern coastline.  The peloton stayed together until the only QOM of the day when Franziska Brauße of Ceratizit-WNT picked up the points meaning she will wear the jersey going into stage 3.  Off the back of Brauße’s move two Italian riders Mathilde Vitillo (Bepink) and Cristina Tonetti (Top Girls Fassa Bortolo) countered a chase group broke away including Inga Češulienê (Aromitalia - Basso Bikes - Vaiano) Beatrice Rossato (Isolmant - Premac - Vittoria) and Marta Jaskulska (Liv Racing Xstra).  With 40 seconds between the two leaders and the chase group and 2:51 back to the peloton the chase group began to close on the pair of Italians in front With 46km to go the two groups joined.   in the interest of bringing Balsamo to the line for a sprint were present on the front of the peloton in an effort to bring back the five leaders however the gap went out to over three minutes the gap started to drop with the efforts in the peloton shaving one minute off the breakaway’s advantage in 10km Jasulska took maximum bonus seconds in the intermediate sprint with 32km to go.   With 20km to go the gap was inside one minute with Jumbo-Visma assisting Trek-Segafredo in the chase in the search for Vos’ 31st Giro Donne stage win As the gap came down to 40 seconds and the peloton started to slow slightly Francesca Pisciali attacked the breakaway but was quickly reeled back The break began to attack one another as the gap tumbled below 30 seconds.  With the breakaway working together and the peloton wary of catching them too soon and inviting counter attacks the gap hovered around 30 seconds with just over 10km to go.  the catch was made and the sprint teams began to line themselves up on the road and Jumbo-Visma were all fighting for position on the road SD Worx started to bring Lotte Kopecky up too.  Movistar were also visible towards the front working for their Danish sprinter Emma Norsgaard Valcar Travel & Service led through the final corner but it was Jumbo-Visma and Trek-Segafredo who were looking strongest in the finishing straight.  Despite the efforts of her Jumbo-Visma squad including Anouska Koster and newly-crowned Dutch National Champion Riejanne Markus Vos was forced to launch her sprint early allowing Balsamo who was brought into position by Elisa Longo Borghini to sit on her wheel until the last minute and come around the Dutch rider to take her first career Giro Donne stage win.  Results powered by FirstCycling The annual celebration of vintage enduro motorcycles from the early 1970s through to the early 1990 the FIM Enduro Vintage Trophy concluded on Sunday MX races following two days of tests and challenges for old bikes (and bones) the final day of competition in Santiago Do Cacem saw all competitors take to the track for the event’s final MX races – just as with the ISDE format and Tulio Pellegrinelli all completed their respective races without incident to claim a commanding margin of victory in the overall Vintage Veterans Trophy Team competition A post shared by enduro21.com (@enduro21_official) “I’ve really enjoyed racing here in Portugal,” said former enduro world champion and Vintage Veterans Trophy Team winner Giorgio Grasso “At the end of the four days we got the win we were looking for The big change we made as a team from previous years was that we came here well prepared in every aspect – we were really ready but at the same time there were some tricky aspects so it’s great to get through the four days with no troubles Team Germany ended the event in second which they too had held since day two of the competition onwards and Sven Roth kept themselves ahead of local favourites Team Portugal and Filipe Guerra who rounded out the Vintage Veterans Trophy Team podium Team Canada earned a well-deserved fourth ahead of Poland All five teams got all three of their riders and their vintage motorcycles to the end of each day of competition with Austria and Spain completing the Vintage Veterans Trophy Team results Italy were also victorious in the Vintage Silver Vase Club Team award with Maurizio Bettini They finished ahead of Spaniards Xavier Soler and David Carrion from Motoclub Cerdanya 1 with MOTO Club Pantera riders Marco Ghilardi At the end of four days of challenging racing 11 Vintage Silver Vase Club Teams managed to get their three riders Topping the Individual class at the 2022 FIM Enduro Vintage Trophy American Fred Hoess ended his four days of racing in Portugal with a highly impressive final day performance setting one of the day’s fastest times during the final MX races Hoess topped the Individual class ahead of Giorgio Volpi Create a personal Enduro21 account to access our new forum receive exclusive competitions and money saving offers Enduro21 is all about motorcycle enduro and off-road riding. Read more Donations to Enduro21 can make a huge difference to what we do Learn more We're on the lookout for writers, photographers, videographers and enduro enthusiasts, from all around the world. Read more This website uses cookies that are necessary to its functioning and required to achieve the purposes illustrated in the privacy policy By accepting this OR scrolling this page OR continuing to browse Metrics details The blood–brain barrier (BBB) poses a significant challenge to drug delivery to the brain A promising approach involves low-frequency low-intensity pulsed ultrasound (US) waves combined with intravenously injected microbubbles (MB) to temporarily and non-invasively open the BBB current technologies cannot easily integrate this procedure with US imaging tracing the harmonic emissions of MB during sonication has been the preferred method for real-time monitoring of US-mediated BBB opening We used an ultrasound advanced open platform (ULA-OP) to simultaneously perform US-mediated BBB opening and US imaging with a single linear-array probe In vitro guinea pig brains were perfused with MB and sonicated with different plane-wave transmission patterns The most effective US pattern was interleaved with B-mode imaging pulses enabling the direct assessment of the MB distribution during treatment The extent of BBB permeabilization was assessed by quantifying FITC-albumin extravasation into the brain via confocal microscopy US-treated hemispheres displayed BBB permeabilization B-mode imaging allowed direct evaluation of MB distribution and interaction with the US beam we achieved effective BBB opening and simultaneous MB imaging using the same diagnostic probe paving the way for US-guided therapeutic ultrasound application in the clinical context The efforts of these authors led to the development of passive acoustic mapping techniques which exploit single or multiple passive cavitation detectors to spatially translate cavitation phenomena and to distinguish stable and inertial cavitation These systems can monitor MB cavitation during sonication allowing for MB spatial localization in the target area at a specific point in time they cannot evaluate MB distribution before and after sonication thus not allowing to study the distribution of MB in the 3D space over time the acoustic feedback from PCD does not consider the real-time variations in the spatial accumulation or flow of intravascular MB among different brain regions and structures which undoubtedly contributes to the bioeffects linked to BBB opening simultaneous US imaging could effectively evaluate the MB 3D distribution during repeated US-induced BBB opening procedures enabling a tailored procedure and achieving the highest accuracy and precision of sonication protocols identifying unique patterns of MB’s distribution across different brain regions could be beneficial to US-mediated BBB opening since different brain areas may display distinct BBB opening patterns due to variations in vascularity and CEUS could be used to identify unique patterns of MB distribution thereby enabling the detection of different patterns of BBB opening by exploiting the intravascular cavitation of MB it is possible to concentrate the mechanical effects of US waves into the vessels This auto-focusing property of MB allows faster and more available treatments US-induced BBB opening might benefit from both an unfocused approach and CEUS-based planning and real-time feedback This approach focused on advanced ultrasound technology to achieve real-time monitoring and precise BBB opening (a) High-resolution confocal microscope photomicrographs of intra-parenchymal FITC-albumin signal in the neocortex following the application of the MB and different US sonication patterns Control brains exhibited only intraluminal signal Patterns B and C yielded predominantly intraluminal signal with dispersed perivascular spots Pattern A and D resulted in more BBB opening as evidenced by areas of FITC-albumin extravasation visible around the vessels (b) and (c) Quantification of FITC-albumin was performed by measuring the area occupied by the fluorescent signal in the control hemispheres and across the four experimental conditions Based on the results of the BBB opening procedure using the four sonication patterns This pattern allowed the introduction of B-Mode imaging sequences between two consecutive treatment sequences thereby enabling direct observation of the interaction between therapeutic US waves and previously infused MB B-mode images showing the MB distribution before (a) The white line represents the base of support for the brain The therapeutic US beam is targeted towards the left hemisphere while the grey diagnostic US beams intersect both hemispheres and fourth panels indicates that the TUS beam is off whereas the red color in the second row indicates that the TUS beam is on (a) B-mode acquisition before the start of US treatment showing the MB distribution within the brain (time t = 0″) (b: t = 80″) and (c: t = 168″) Effects of the interfering TUS beam on MB distribution during and at the end of the US treatment (d: t = 180″) B-mode acquisition following the conclusion of the US treatment we have demonstrated the feasibility of disrupting the BBB while concurrently performing B-mode imaging through the use of a single clinical diagnostic probe focusing only on the occurrence of cavitation without evaluating MB distribution across time and space Real-time CEUS imaging could overcome this limitation by assessing 3D MB distribution in target tissues during repeated therapeutic BBB procedures This would allow tailored sonication parameters to minimize the impact on physiological structures patterns A and D were more effective than patterns B-C This is due to the higher PRF (2 kHz) employed in A and D which is needed to obtain adequate BBB-opening when working with low-pressure pulsed US (340 kPa) The most effective transmission pattern in terms of BBB opening (pattern D) was interleaved with the transmission of B-mode imaging sequences This pattern was employed to observe the interaction between MB and TUS waves and the effect that sonication has on MB distribution a reduction in MB signal intensity was observed in the treated area This reduction can be attributed to the acoustic cavitation phenomenon We believe that at least some MB collapse due to transient – not sustained—inertial cavitation caused by the pressures used by our ultrasound device the negative effects of sustained inertial cavitation were not observed in our model This observation is a crucial step to unveil MB dynamics during TUS procedures and was possible thanks to the concomitant US imaging provided by our system The B-mode modality of the system demonstrated a persistent reduction in MB signals even after the US treatment This probably indicates persistent cerebral hypoperfusion due to cerebral vasospasm with the restoration of the initial MB distribution pattern thereby confirming the reversibility of the BBB opening procedure as sonication pattern D3 was used solely in the final experiment further trials should be conducted with this pattern to verify the reproducibility of the results and to confirm the efficacy of B-mode imaging during BBB opening procedures This study provides evidence of the effectiveness of the ULA-OP system in real-time monitoring BBB opening enabling both US imaging and US disruption treatment to be performed simultaneously The anatomy of the brain and the circulation of MB were easily visualized through US imaging and it was possible to observe real-time changes in the B-mode pattern during US treatment This is of great relevance as it would allow us to adjust sonication parameters to align with the pattern of MB enhancement of target tissues A significant aspect of this study is that the US probe was the same as those used in current clinical practice possibly smoothening the transition between the pre-clinical and the clinical setting Trans-prostheses BBB-opening would be performed post-operatively in a selected cohort of patients that require cranial surgery our approach does not increase the invasiveness of the standard of care and would allow us to follow-up patients with trans-prostheses US imaging and therapy in an out-patient setting The results of the present study represent a significant advance in the field of ultrasound-mediated BBB opening overcoming the limitations of passive cavitation detectors Our system exploits the ambivalent nature of MB which can be used for both imaging and therapy by using a single US probe capable of performing both TUS and imaging simultaneously This approach allows for the direct assessment of MB circulation within the treated area enabling real-time spatial imaging of TUS-induced BBB opening 16 hemispheres from 8 guinea pig brains were sonicated with the ULA-OP US system were programmed to understand which modality yields the best results in terms of BBB opening one hemisphere was sonicated with a modified version of pattern D whereas the contralateral served as control 16 mm-wide plane waves were generated by 64 adjacent elements of the linear array probe through the transmission of 4 MHz sinusoidal pulses with programmable length (T) and pulse repetition frequency (PRF) The amplitude of these pulses produced a peak-negative-pressure (PNP) of 340 kPa in water The total duration of the treatment interval was always 120″ approximately 0.5″ of sonication distributed over such an interval As reported in Table 2 and C were designed to produce stimulation with PRF of 2 kHz and 80 Hz and correspondingly increasing pulse durations so that a duty cycle (T × PRF) of 0.4% was maintained before being repeated until the 120 s treatment interval was fully covered The pattern labeled D3 was derived from pattern D by inserting the transmission of B-Mode imaging sequences during the 480 ms of transmission pause transmitting 144 pulses (3-cycle at 5 MHz) allowed to achieve an overall rate of 4 frames/s The experimental procedures and the animal care were conducted following the guidelines defined by the European Communities Council directive (2010/63/EU).) Every effort was made to limit the number of animals used The experimental protocol was reviewed and approved by the Ethics Committee and by the Committee on Animal Care and Use of the Fondazione IRCCS Istituto Neurologico Carlo Besta (Approval reference number: DO-01-2021) the guinea pig is transcardially perfused using a peristaltic pump with a cold (15 °C) oxygenated (95% O2 / 5% CO2; pH 7.1) complex saline solution composed by NaCl and the brain is carefully transferred into the incubation chamber whose bottom is coated with a US-absorbing plate The brain’s ventral surface is positioned upward to visualize the base of the brain and the olfactory bulbs and the cervical spinal cord are held down by two silk threads for mechanical stabilization a polyethylene cannula (terminal gauge 0.25 mm) is inserted into the basilar artery to allow the restoration of brain circulation with the above solution (pH 7.3) at a rate of 6 ml/min via a peristaltic pump (Gilson Minipulse 4) the carotid and the hypophyseal arteries are closed to re-establish the physiological perfusion of the brain The temperature of the incubation chamber is successively increased from 15 to 22 °C Isolated in vitro guinea pig brain can maintain close to-physiological activity for approximately 6–8 h Scheme of the experimental setup for MB perfusion via the resident arterial system of the in vitro isolated brain preparation. MB are injected via a syringe tributary of the main perfusion line. MB sonication was performed using the LA332 probe connected to the ULA-OP platform and oriented in the coronal plane over the brain The area of influence of the TUS beam was limited to one hemisphere A US-absorbing polyurethane plate was positioned at the bottom of the incubation chamber to prevent US wave reflections The extravasation of fluorescein isothiocyanate (FITC)-albumin (50 mg/10 ml perfused for 4 min at the end of each experiment was quantified using a confocal microscopy to evaluate US-mediated BBB opening Brains were removed from the incubation chamber after each experiment and were fixed by immersion for 48 h in a cold 4% paraformaldehyde solution in phosphate-buffered saline (PBS; 0.1 M Serial coronal sections were obtained by vibratome (VT 1000S Leica) throughout the extension of the hippocampus (plates A5.4–A6.24 of the guinea pig brain atlas by Luparello); sections were then collected on gelatin-coated slides mounted in Fluorosave (Calbiochem) and cover-slipped Two representative sections corresponding to coronal rostral-caudal levels A5.40 and A6.00 of the Luparello atlas were collected for each brain Slices were studied using a laser scanning confocal microscope D-Eclipse C1 (Nikon) mounted on a light microscope Eclipse TE2000-E (Nikon) using excitation light of 488 nm (Laser Ar) The FITC-albumin fluorescent signal was quantified in the neocortex Five high-power non-overlapping fields (region of interest [ROI] of 1.6 mm2 each) per section were acquired bilaterally at × 10g The Image-Pro Premier 9.1 software (Media Cybernetics) was used to assess and quantify the percentage of FITC-albumin signal (number of pixels) in each nonoverlapping field and the data measured per slice were finally averaged providing a single value for each hemisphere The normal distribution of samples was checked with the Shapiro-Wilks test and the homogeneity of variances was evaluated with the F test The nonparametric Mann–Whitney tests was used The format of Mann–Whitney test results is: median; U = x All statistical tests were performed with Origin 9.0 (OriginLab Corporation) The tests are two-sided and a confidence interval (CI) of 95% (p ≤ 0.05) was required for values to be statistically significant The data illustrated with boxplots show median (central line) The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request Daneman, R. & Prat, A. The blood–brain barrier. Cold Spring Harbor Perspect. Biol. 7(1), a020412. https://doi.org/10.1101/cshperspect.a020412 (2015) Alahmari, A. Blood-brain barrier overview: Structural and functional correlation. Neural Plast. 2021, 1–10. https://doi.org/10.1155/2021/6564585 (2021) Kadry, H., Noorani, B. & Cucullo, L. A blood–brain barrier overview on structure, function, impairment, and biomarkers of integrity. Fluids Barriers CNS https://doi.org/10.1186/s12987-020-00230-3 (2020) Ultrasound-induced blood-brain-barrier opening enhances anticancer efficacy in the treatment of glioblastoma: Current status and future prospects Helfield, B. A review of phospholipid encapsulated ultrasound contrast agent microbubble physics. Ultrasound Med. Biol. 45, 282–300. https://doi.org/10.1016/j.ultrasmedbio.2018.09.020 (2019) transcranial and localized opening of the blood-brain barrier using focused ultrasound in mice Padilla, F., Brenner, J., Prada, F. & Klibanov, A. L. Theranostics in the vasculature: Bioeffects of ultrasound and microbubbles to induce vascular shutdown. Theranostics 13(12), 4079–4101. https://doi.org/10.7150/thno.70372 (2023) Burgess, A., Shah, K., Hough, O. & Hynynen, K. Focused ultrasound-mediated drug delivery through the blood–brain barrier. Expert Rev. Neurother. 15(5), 477–491. https://doi.org/10.1586/14737175.2015.1028369 (2015) A three-dimensional model of an ultrasound contrast agent gas bubble and its mechanical effects on microvessels Aryal, M., Arvanitis, C. D., Alexander, P. M. & McDannold, N. Ultrasound-mediated blood–brain barrier disruption for targeted drug delivery in the central nervous system. Adv. Drug Deliv. Rev. 72, 94–109. https://doi.org/10.1016/j.addr.2014.01.008 (2014) Carpentier, A. et al. Clinical trial of blood-brain barrier disruption by pulsed ultrasound. Sci. Transl. 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Methods 260, 83–90. https://doi.org/10.1016/j.jneumeth.2015.03.026 (2016) Blood-brain barrier preservation in the in vitro isolated guinea pig brain preparation A reconfigurable and programmable FPGA-based system for nonstandard ultrasound methods Download references Part of the activities were granted through fellowships provided by the Focused Ultrasound Foundation We would like to thank the Ultrasound Lab of the Istituto Nazionale di Ricerca Metrologica (INRiM) Italy and the Department of Information [FP1] Engineering Italy for sharing technical materials and advice Acoustic Neuroimaging and Therapy Laboratory (ANTY-Lab) Nicoletta Corradino & Francesco DiMeco Fondazione IRCCS Istituto Neurologico Carlo Besta Istituto Nazionale di Ricerca Metrologica I.N.Ri.M. designed and supervised the study and reviewed the manuscript; P.T provided ULA-OP system and parameters during all experiments; F.P. performed insonations and ultrasonographic acquisitions provided the isolated brain preparations and the perfusion system and performed statistical analyses on BBB opening; L.L performed histological preparation of the specimens acquired and analyzed the images at confocal microscopy; F.P. The authors declare no competing interests Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41598-025-94660-4 Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Sign up for the Nature Briefing newsletter — what matters in science Any hard core ISDE and World Enduro fan will certainly recognize the names Giorgio Grasso Tulio Pellegrinelli and Enrico Tortoli who were the winners of this year’s FIM Enduro Vintage Trophy Team Championship in Portugal The trio made up the winning Italian squad in this four-day ISDE format competition Team Italy went into the final MX test on Sunday with the lead and held on for the overall victory against more than 130 of the sport’s top veteran off-road racers that participated “I’ve really enjoyed racing here in Portugal,” former Enduro World Champion Grasso said “At the end of the four days we got the win we were looking for The big change we made as a team from previous years was that we came here well prepared in every aspect—we were really ready so it’s great to get through the four days with no troubles it was the lone American who captured the overall victory ISDE veteran and multi-time gold medalist Fred Hoess was the top-finishing rider overall was the top rider on day three and had one of the day’s fastest times in the MX test Second overall went to Giorgio Volpi and third to Adriano Micozzi Clocking two laps of a 48.5 kilometre course with two special tests per lap day three was shorter but still tough enough to make sure riders were challenged on Spanish Pyrenees trails Carrying a reasonably comfortable lead after this last day of the enduro tests defending EVT champions Team Italy head France and Germany with Tem USA fourth and Portugal completing the top five may be familiar to anyone who was riding and racing when these various vintage bikes were new The FIM Enduro Vintage Trophy (EVT) brings together riders from all over the world with legends of the sport popping up inside the tests including one of the best female riders on the planet Mireia Badia turning her hand to vintage enduro Another rider winding back the clock and enjoying racing in Spain is American team rider Lendon Smith As the owner of seat manufacturer Seat Concepts Lendon has seen his passion for older motorcycles grow into a business “Around 2004 I started making seat for vintage bikes It just about paid enough for myself and some friends to go racing at that time Around 2009 I got more serious with the business and I decided to try and make seat for more bikes things took off and from dual sport and adventure we moved into competition motorcycle seats and today we have 46 employees Things have worked out pretty well.” Lendon (right) is enjoying his racing more than ever “I didn’t get into dirt bikes until I was 14 but by the time I was 18 I was Florida state Hare Scramble champion and business kind of curtailed my motorcycle racing I told my wife I wanted to start racing again And that’s kinda lead to me racing here at the EVT “I used to do a lot of vintage motocross racing But this is my first EVT and probably my first vintage race in 12 years I wasn’t expecting the trails to be as good as they are They’re incredible and the scenery has been just amazing so it’s taken me a few days to get to grips with it Day three has been great for me and I can’t wait for the final day motocross races.” is the traditional MX taking place at the Sant Marc circuit which will be followed by Prize Giving Ceremony More information and live results: www.cronooffroad.com www.motoclubcerdanya.com Follow @fim_enduro_vintage on Instagram for daily stories direct from the EVT Photo Credit: Future7Media | Nicki Martinez © Enduro21 / Future7Media Limited. 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Volume 15 - 2024 | https://doi.org/10.3389/fimmu.2024.1355764 Skin and soft tissue infections (SSTIs) are the most common diseases caused by Staphylococcus aureus (S which can progress to threatening conditions due to recurrences and systemic complications Staphylococcal protein A (SpA) is an immunomodulator antigen of S which allows bacterial evasion from the immune system by interfering with different types of immune responses to pathogen antigens Immunization with SpA could potentially unmask the pathogen to the immune system leading to the production of antibodies that can protect from a second encounter with S as it occurs in skin infection recurrences we describe a study in which mice are immunized with a mutated form of SpA mixed with the Adjuvant System 01 (SpAmut/AS01) before a primary S Although mice are not protected from the infection under these conditions they are able to mount a broader pathogen-specific functional immune response that results in protection against systemic dissemination of bacteria following an S We show that this “hidden effect” of SpA can be partially explained by higher functionality of induced anti-SpA antibodies a broader and stronger humoral response is elicited against several S aureus antigens that during an infection are masked by SpA activity aureus spreading from the skin through the blood the development of a broader immune response after immunization with SpA and infection with S aureus could represent an effective protection against a second exposure to the pathogen To understand if the immunomodulator properties of SpA could affect the humoral response after an S we used a mouse model of skin infection and recurrence and characterized the humoral response in animals vaccinated with SpAmut aureus USA300 LAC strain was used for the in vivo model of skin infection and recurrence Bacteria were grown in tryptic soy broth (TSB) at 37°C in agitation until the early exponential phase is reached and diluted 1:1 in a freezing solution composed of phosphate-buffered saline (PBS) + 10% bovine serum albumin (BSA) + 10% L-glutamic acid Aliquots were conserved at −80°C in cryovials until use 2-mL frozen stocks of bacteria were thawed and diluted in 48 mL of fresh TSB and incubated at 37°C at 250 rpm until bacteria reached the early exponential phase (OD600 = 2.0) at approximately 1.5–2 × 109 colony-forming units (CFUs)/mL Bacteria were then washed once in sterile PBS and diluted with sterile PBS to obtain 4 × 108 CFUs/mL or 8 × 108 CFUs/mL for the skin infection or recurrence models aureus USA300 LAC strain expressing the green fluorescent protein (GFP) was used for the internalization assays Bacteria were streaked on a tryptic soy agar (TSA) plate and incubated overnight (O/N) at 37°C + 5% CO2 pre-inocula were prepared from single colonies and they were incubated O/N at 37°C at 250 rpm the cultures were diluted in fresh TSB and incubated at 37°C and 250 rpm for approximately 3 h until they reached the early exponential phase (OD600 = 2) Bacteria were centrifuged at 2,400 rpm and 4°C for 10 min and washed twice with RPMI 1640 GlutaMAX + 25 mM HEPES + 0.05% BSA the OD600 of bacteria was measured and adjusted to an OD600 of 1.0 Bacteria were aliquoted in 2-mL cryovials and stored at −20°C until use Animal husbandry and experimental procedures were ethically reviewed and carried out in accordance with European Directive 2010/63/EU and GSK Vaccines’ Policy on the Care and were approved by the Italian Ministry of Health (authorization 123/2015-PR) Animals were kept in an AAALAC-accredited facility animals were randomly distributed in different experimental groups in individually ventilated cages (IVC The acclimation lasted for a period of 5 days each animal was identified by an individual tattoo All animals had ad libitum access to GMP-grade food (Mucedola 4RF25 TOP CERTIFICATE) and bottled irradiated cellulose bags containing Mucedola SCOBIS UNO bedding and carboard tunnels (ANTRUM) or plexiglass mouse houses were provided within the cages A few food pellets in the cage were also used as enrichment for foraging and additional gnawing Cage and bedding changes were performed once every 2 weeks in agreement with the cage supplier’s indications Air supplied in IVC was 100% fresh air filtered by an EPA filter via the IVC system The animal room conditions were as follows: temperature 21°C (± 3°C); relative humidity 50% (range 30%–70%); and 12-h light/12-h dark cycle and relative humidity were recorded continuously by room probes while the IVC system recorded the individual motors’ performance The light cycle setting was ensured by a validated alarm system The mouse model of skin infection and recurrence was performed as depicted in the Supplementary Materials (see Supplementary Material Figure S1) Five-week-old female specific pathogen-free (SPF) C57BL/6 mice were immunized twice intramuscularly (IM) Animals were immunized with 10 µg of a SpAmut adjuvanted with AS01 The control group (sham) was immunized with the formulation buffer alone (10 mM Na2HPO4 and 150 mM NaCl the back of mice was shaved under anesthesia (3.5% isoflurane) using an electric razor and depilatory cream mice were inoculated subcutaneously (SC) in the right flank with 2 × 107 CFU/50 µL of S mice were shaved and infected again as previously described in the opposite flank with 4 × 107 CFU/50 µL of S aureus USA300 LAC strain to establish the model of skin infection recurrence blood was withdrawn from mice and serum was collected by centrifugation and skin biopsies and kidneys were collected serially diluted and plated for bacterial burden and expressed as CFU/mL for skin biopsies or as dissemination index for kidneys 20 µg of recombinant SpAWT or SpAmut was chemically coupled to 1.25 × 106 MagPlex beads (Luminex Corporation) through an automated coupling method with a liquid handling workstation (Hamilton – Microlab STAR IVD) antigens were coupled by a two-step carbodiimide procedure during which microsphere carboxyl groups are first activated with 1-ethyl-3-(3-dimethylaminopropryl) carbodiimide hydrochloride (EDC in the presence of sulfo-NHS (Pierce) to form a sulfo-NHS-ester intermediate The reactive intermediate is then replaced by a reaction with the primary amine of the target molecule to form a covalent amide bond Total SpAmut-specific IgG titers were evaluated with an automatized Luminex assay incubating serially diluted mice sera with SpAmut-coupled beads for 1 h with vigorous shaking IgG titers were detected using an R-Phycoerythrin AffiniPure F(ab′)2 Goat anti-mouse IgG (Jackson ImmunoResearch Labs RRID: AB_2338627) through a FLEXMAP 3D reader (Luminex Corporation) Median fluorescence intensities (MFIs) of the samples subtracted to the MFI of the blanks (MFI-Bkgd) expressing the IgG titer as relative light units per milliliter (RLU/mL) the experiment was considered valid if a titer was observed in at least three points of the dilution and if these titers were interpolated in the linear portion of the standard curve; the final IgG titer of each value was expressed as the median value of the observed titers threefold serially diluted pools of sera collected after the second dose of immunization (D52 2 weeks post-infection) were incubated with SpAmut-coupled beads for 1 h with vigorous shaking repeating the assay in five different 96-well plates the specific IgG subclass was detected using the appropriate R-Phycoerythrin AffiniPure F(ab′)2 Goat anti-mouse IgG (Cat #115-116-072 RRID : AB_2338624) secondary antibody (Jackson ImmunoResearch Labs) Results were detected using a Luminex 200 instrument system (Luminex Corporation) and they were reported as the ratio between the RLU/mL of the different IgG subclasses a mouse standard serum and mice sera obtained after immunization (D52 2 weeks post-infection) were threefold serially diluted and incubated in duplicate with SpAmut-coupled beads for 1 h with vigorous shaking one-half of the samples was incubated with vigorous shaking either with 2 M ammonium thiocyanate or with PBS for 30 min the remaining IgGs were detected with an R-Phycoerythrin AffiniPure F(ab′)2 Goat anti-mouse IgG (Jackson ImmunoResearch Labs) using a Luminex 200 reader (Luminex corporation) Results were reported as avidity percentage calculated on the basis of the ratio of the antibody titers recovered after incubation with ammonium thiocyanate with respect to titers observed after incubation with PBS The displacement assay was performed as described elsewhere (17) SpAWT-coupled beads (prepared as already described above) were saturated with 10 µg/mL of biotinylated-human IgGs for 30 min under vigorous shaking saturated beads were incubated with threefold serially diluted total IgGs purified from mice sera collected after immunization (D52 2 weeks post-infection) or with commercial total mouse IgGs as negative control (ChromPure Mouse IgG the amount of biotinylated-human IgGs bound to the SpAWT-coupled beads was detected using 10 µg/mL of Streptavidin Results are shown as the percentage of antibody displaced normalized on the signal obtained without adding mouse IgGs Slides were conserved in the dark at 4°C until use After saturating the slides with a blocking buffer (BlockIt diluted sera samples (n = 9) obtained 2 weeks after infection from immunized (SpAmut/AS01 and infected) or non-immunized (sham and infected) mice were incubated on slides for 1 h in the dark in a humid chamber signals were detected with an Alexa Flour 647 anti-mouse IgG secondary antibody (Jackson ImmunoResearch Labs RRID : AB_2338903) and slides were rinsed and left to dry Slides were scanned using an InnoScan 710 AL (INNOPSYS) and images were generated by the Mapix Microarray Image Analysis Software Fluorescence intensities of spots were determined using ImaGene 9.0 software (Biodiscovery Inc.) and data analysis was performed using R scripts Each spotted antigen was considered as “positive” if recognized by the majority of tested sera (at least five out of nine) THP-1 cells (ATCC TIB-202, RRID: CVCL_0006) were cultured in RPMI 1640 medium supplemented with GlutaMAX and HEPES (Gibco), 10% FBS, and 5 μg/mL of penicillin/streptomycin, according to manufacturer indications. Single sera collected from animal studies were heat-inactivated at 56°C for 30 min. The opsonophagocytosis assay was performed as described elsewhere (17) aureus USA300 GFP strain were pre-opsonized with mouse sera obtained from normal mice (Pre-immune) 2 weeks after infection from non-immunized mice (sham and infected) and immunized mice (SpAmut/AS01 and infected) for 30 min at 37°C under vigorous shaking A total of 3–4 × 104 THP-1 cells were added to allow opsonization of bacteria and incubation continued for an additional 30 min (bacteria-to-cell ratio of 200:1) After adding lysostaphin (Sigma-Aldrich) to lyse non-internalized bacteria bacteria and cells were fixed with 2% formaldehyde for 1 h on ice prior to acquisition at FACS CANTO II (Becton Dickinson) and data collected were analyzed using the FACSDiva 8.0.1 software Gating strategy was as follows: (i) THP-1 cells; (ii) singlet cells; and (iii) FITC-A representative of cells that have effectively internalized GFP-expressing bacteria The median percentages of GFP+ THP-1 cells were normalized on the median signal obtained with pre-immune sera and results were then represented as fold change with respect to the median of pre-immune sera A p (value) ≤ 0.05 was considered significant for all these analyses Legend: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001; ns = not significant Figure 1 Subcutaneous infection with S aureus does not induce anti-SpAmutIgG in mice Anti-SpAmut IgG titration expressed as relative light units (RLU) per milliliter in sera of animals only immunized or post-infection (D72) sera were analyzed and titers against SpAmut were reported in the graph while SpAmut/AS01 animals were immunized with the proposed vaccine The median value and the 95% confidence interval were reported for each group and condition Groups were represented by at least 7 animals (range The Kruskal–Wallis and uncorrected Dunn’s posttest was used to assess significance Figure 2 Vaccination with SpAmut/AS01 protects against local manifestations and systemic sequelae in a skin infection recurrence mouse model of S B) and systemic (C) readouts used to assess protection of SpAmut/AS01 vaccine in the skin infection recurrence model (A) Area under the curve (AUC) indicated as mm2 of dermonecrotic lesion developed from day 4 to day 7 after skin infection recurrence The red dotted line represents the Lower Detectable Value (LDV) (B) Log10 of colony-forming unit (CFU) counts enumerated in homogenized skin biopsies collected 7 days after skin infection recurrence (C) Dissemination severity index assigned to single mice based on CFU counts recovered in the kidneys of infected mice 7 days after skin infection recurrence very low dissemination (1–10 CFU/kidneys); Score 2 minimal dissemination (11–100 CFU/kidneys); Score 3 mild dissemination (101–1,000 CFU/kidneys); Score 4 severe dissemination (>1,001 CFU/kidneys) ***pval< 0.001; ****pval< 0.0001; n.s each single dot represented data from a single animal and red lines were median values of the groups A total of 29 animals belong to the age-paired naïve mice (Naïve) group 18 animals make up the group of mice not vaccinated but exposed to S aureus during the first infection (sham and infected) and 18 animals belong to the group of mice vaccinated and infected (SpAmut/AS01 and Infected) The Kruskal–Wallis and uncorrected Dunn’s post-test was used to assess significance among groups Figure 3 Antibodies in sera from SpAmut/AS01 and infected mice increase S aureus phagocytosis by specialized human cells Percentage of human THP-1 monocytic cells that internalized GFP-labeled S Box-and-whiskers representation of THP-1 cells that have internalized bacteria Lines delimiting the boxes reported the median value of the group with 25 and 75 percentiles The whiskers reported 10 and 90 percentiles Eight pools (from at least five mouse sera for each group) were used for the assay The Kruskal–Wallis and uncorrected Dunn’s post-test was used to assess significance Figure 4 Vaccination with SpAmut/AS01 expands the antibody repertoire following an S Nine single sera obtained from immunized and infected (SpAmut/AS01 and infected) and from infected-only animals (sham and infected) were assessed against the staphylococcal protein microarray Out of the 96 antigens spotted on the array 23 reacted with higher frequency with SpAmut/AS01 and infected sera with respect to sera obtained from infected-only animals Filled box: reactive serum; non-filled box: non-reactive serum GAG L: Glycosaminoglycan lyase; RDG-lipop.: RDG lipoprotein; Thermon: thermonuclease *pval< 0.05; **pval< 0.01; ***pval< 0.001; ****pval< 0.0001 Fisher’s exact t-test was used to evaluate significant differences between recognition frequencies for each antigen The area under the curve (AUC) was used to perform a cumulative data analysis on the dermonecrotic lesion areas (Figure 2A) The trapezoidal AUC of the dermonecrotic lesion areas was calculated using the following formula: The AUC was calculated for each time point after infection in the single animal and eventually summed together to have a final AUC value representing the whole time period after the infection Vaccination with SpAmut was shown to protect guinea pigs against systemic disease and to enable the host to develop a broader antibody repertoire against several staphylococcal antigens otherwise masked by SpA activity, which could protect from a second encounter with S. aureus (8) we developed an animal model to evaluate the contribution of SpA-specific antibodies in mediating the protection to systemic sequelae of skin infection recurrences Different readouts were used to monitor the effect of vaccination and of the infection Results were compared with those obtained with mock immunized mice We also evaluated a possible effect of immunization with the proposed vaccine in a model of skin infection but no reduction in dermonecrotic area size and CFU counts either in the skin or in the kidneys as observed (see Supplementary Material Figure 2) Immunization with SpAmut/AS01 expanded the immune response to the pathogen after infection of mice which overall contributed to control local disease (bacterial load in skin lesions) and bacterial dissemination to kidneys in a skin infection recurrence model We then wondered whether a change in the quality of anti-SpAmut IgG immune response could be detected after infection. IgG subclasses of anti-SpAmut specific antibodies were therefore analyzed in mouse sera, showing that no significant changes in the antibodies repertoire occurred during infection (Table 1) affinity of anti-SpAmut IgG was characterized in sera collected from mice before and after subcutaneous infection anti-SpAmut antibodies strongly increased in affinity for their target during infection with a relative percentage of IgG bound to the protein in the presence of 2 M ammonium thiocyanate that passed from 28% to 84% Table 1 SpAmut-specific IgG subclasses composition analysis after immunization and infection This effect was strongly enhanced when IgG purified from animals immunized and then infected were used with a 60% IgG displacement and an increase of threefold as compared to the negative control aureus infection increases the ability of vaccination to induce antibodies that allow in vitro human IgG displacement from SpA wild-type protein Percentage of human IgG displaced from SpAWT-coupled beads by purified IgGs from sera of animals only immunized with SpAmut/AS01 or immunized and infected animals Results were expressed as the percentage of human antibody displaced from the SpAWT Table 2 List of the 23 antigens that reacted with higher frequency with sera from immunized and infected mice Sera of SpAmut/AS01 and infected mice, as compared to sera from only vaccinated or sham and infected mice, elicited antibodies that (i) showed an increased affinity for SpA,(ii) reduced SpA ability to bind immunoglobulins (Figure 5), and (iii) recognized more S. aureus virulence factors both secreted and exposed on the bacterial surface (Figure 4; Table 2) Antibodies present in sera of these animals before the second subcutaneous infection (recurrence) could therefore facilitate the clearance of bacteria aureus from skin to kidneys was highly impaired in SpAmut/AS01-immunized and infected animals To further evaluate the contribution of SpA-specific antibodies in the elimination of S. aureus from the blood circulation, a human monocytic cell line (THP-1) was used to evaluate bacterial internalization in the presence of sera from different groups of mice. As reported in Figure 3 neither vaccination of mice with SpAmut/AS01 (fold change equal to 0.7 with respect to the pre-immune sera and 90% percentile was 1.18) nor bacterial infection (fold change equal to 3 with respect to the pre-immune sera and 90% percentile was 5.8) significantly enhanced phagocytosis as compared to the negative control the combination of vaccination and infection generated sera that significantly increased the phagocytic properties of THP-1 cells compared to the negative control (fold change equal to 5 also to vaccination alone (pval< 0.0001) we have demonstrated that the efficacy of a vaccine candidate especially if it contains an immunomodulator virulence factor could go beyond a direct effect of antibodies and cellular response against the antigen itself A detoxified version of Staphylococcal protein A (SpAmut) triggered the mouse immune system to mount a broader and more mature serological response against S aureus during a first encounter with the pathogen which importantly protected the animals against a second infection Considering that skin infection and recurrences are one of the most frequent S the results obtained in an animal model somehow mimicking the human disease are particularly relevant and this can have an impact on the immune response elicited by a subsequent vaccination and on the effect mediated by SpA (“indirect” protection mediated by SpA-antibodies) Follow-up studies should be done to further address this point aureus antibodies is impaired under these conditions aureus are therefore often inefficient in clearing the infection Recurrences and bloodstream dissemination, both strictly connected to the ability of S. aureus to hide in the immune system, are the hallmark of SSTIs and exemplify the inability of infected hosts to establish a protective immunity (1, 38, 39) a mouse model of skin infection and recurrence has been chosen to (i) evaluate whether immunization with SpA could unmask the pathogen also in a model of infection that resembled the most frequent staphylococcal disease in humans; (ii) measure the impact that this unmasking effect could have against a future encounter with bacteria in such a model Vaccination with SpAmut/AS01 did not directly protect animals against skin infections and related sequelae but triggered the host to mount a protective immune response during this first encounter, which proved to have a crucial importance for a second infection (Figure 2; see also Supplementary Figure 1) the immunization with the proposed vaccine contributed not only to increase the number of S aureus virulence factors recognized during infection but also to improve the quality of the mounted immune response aureus to use its elusive properties when reinfecting the host the control of systemic sequelae probably needed a more diverse and complete immune response like that developed by vaccinated and infected mice Interestingly, since human neutrophils utilize the same FcγRs to trigger phagocytosis, we could hypothesize that this mechanism of protection could also be extended in humans during a systemic spreading of S. aureus, even if in vitro phagocytosis performed with a human monocytic cell line could not directly correlate with a possible protection in vaccinated people (44–47) To conclude, even if we could not exclude the idea that the cellular response may have had a partial contribution on the overall observed protection, IgG-mediated opsonophagocytosis is certainly one of the most important mechanisms of bacterial clearance that could explain our results (16, 48) These data strongly support the proposal to use this antigen in a vaccine against S and it would be better if it was combined with additional antigens that could offer direct protection during a first encounter with the pathogen The original contributions presented in the study are included in the article/Supplementary Material Further inquiries can be directed to the corresponding author The animal study was approved by Italian Ministry of Health (authorization 123/2015-PR) The study was conducted in accordance with the local legislation and institutional requirements The author(s) declare that no financial support was received for the research We thank Elena Boero for the support on the opsonophagocytosis assay Viola Viviani per the protein microarray and related data analysis Patrizia Giannetti for vaccine formulation Fabiana Spensieri and Stefano Bonacci for supporting with all the Luminex analyses A special acknowledgment is due to Raffaella Cecchi Tommaso Pasquali and all the GSK Animal Resources Center Italy for their excellent support with the in vivo studies We also thank Professor Chiara Falciani for all the precious support and critically revising data AM was a PhD student of Università degli Studi di Siena Italy at the time of the study and supervised by GSK GM was a student of Università di Bologna Italy at the time of the study and participated in an internship at GSK a contract employment organization contracted by GSK DL and GB were employee of GSK at the time of the study FB is listed as inventor on patents concerning S The author(s) declared that they were an editorial board member of Frontiers This had no impact on the peer review process and the final decision All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2024.1355764/full#supplementary-material Staphylococcus aureus infections: epidemiology Co-evolutionary aspects of human colonisation and infection by Staphylococcus aureus Strategies for and advances in the development of Staphylococcus aureus prophylactic vaccines Staphylococcus 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protein A protects mice against systemic complications of skin infection recurrences Received: 14 December 2023; Accepted: 16 February 2024;Published: 11 March 2024 Copyright © 2024 Mandelli, Magri, Tortoli, Torricelli, Laera, Bagnoli, Finco, Bensi, Brazzoli and Chiarot. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) distribution or reproduction in other forums is permitted provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited in accordance with accepted academic practice distribution or reproduction is permitted which does not comply with these terms *Correspondence: Emiliano Chiarot, ZW1pbGlhbm8ueC5jaGlhcm90QGdzay5jb20= Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher 94% of researchers rate our articles as excellent or goodLearn more about the work of our research integrity team to safeguard the quality of each article we publish Italy – Andre Greipel of Germany won a bunch sprint to take the second stage of the Giro d’Italia on Saturday and the pink jersey for the first time Greipel edged out Roberto Ferrari at the end of the hilly 221-kilometer (137-mile) leg from Olbia to Tortoli Jasper Stuyven was third on another slow day at the Giro with lots of crosswinds and some headwinds It made a six-hours race but it also played in our favor,” said Greipel “Otherwise there would have been some attacks “I’m really proud to win at the Giro again This win and the pink jersey is for (my teammates) It appeared as if it would be a three-way sprint between Caleb Ewan but Ewan’s foot slipped out of the pedal after rubbing shoulders with Gaviria The third stage on Sunday is a 148-kilometer (92-mile) route from Tortoli to Cagliari Monday is a rest day and the transfer to Sicily Give directly to The Spokesman-Review's Northwest Passages community forums series -- which helps to offset the costs of several reporter and editor positions at the newspaper -- by using the easy options below Gifts processed in this system are tax deductible Get the day’s top sports headlines and breaking news delivered to your inbox by subscribing here © Copyright 2025, The Spokesman-Review | Community Guidelines | Terms of Service | Privacy Policy | Copyright Policy 'I need to trust myself more' says Team DSM rider however she found herself out of position and Wiebes reacted to take the victory herself.  This time, the team’s focus was all for Kool going into the sprint on stage 2 of the Giro d’Italia Donne The 23-year-old looked strong but was boxed in at a crucial moment she still made a strong comeback and came fast towards the line to take third place behind winner Elisa Balsamo (Trek-Segafredo) and second-placed Marianne Vos (Jumbo-Visma) The 106.4km stage into Tortolì looked destined to come down to a bunch kick with a breakaway being allowed over three minutes before the peloton reeled them in with around 10km to go.  “It was a pretty easy but warm day,” Kool said after the race. “The team did really well having a lot of bottle points for us so we could stay really cool in the end which helped me get over the heat.  "We really stuck to the plan throughout the day Leah [Kirchmann] and Franzi [Franziska Koch] positioned us like we had talked about for every important moment I lost Franzi a little bit because we were stuck behind the others I had a strong sprint and came really close in the end so I think I need to trust myself more and I could maybe have started sprinting earlier I was a bit boxed in and had to come late but we hope to do better tomorrow and aim for that win.” Kool has four more potential chances at sprinting to victory in this year’s race depending on the fate of any possible breakaways and 10 all presenting possibilities for sprint finishes.  “We did a really good job to bring Charlotte in a good position for the final bend and in the end she got third place It’s a podium which is great but we’re motivated to go for even more in the next days,” said Team DSM coach Huub Duijn Metrics details Mycobacterium tuberculosis and Mycobacterium leprae have remained the primary species of the genus Mycobacterium of clinical and microbiological interest referred to as nontuberculous mycobacteria (NTM) the number of reports linking various NTM species with human diseases has steadily increased and treatment difficulties have emerged Despite the availability of whole genome sequencing technologies limited effort has been devoted to the genetic characterization of NTM species the taxonomic and phylogenetic structure of the genus remains unsettled and genomic information is lacking to support the identification of these organisms in a clinical setting we widen the knowledge of NTMs by reconstructing and analyzing the genomes of 41 previously uncharacterized NTM species We provide the first comprehensive characterization of the genomic diversity of NTMs and open new venues for the clinical identification of opportunistic pathogens from this genus are widespread or not in the many NTM species without available genomic information Given the current availability and cost-effectiveness of whole-genome sequencing (WGS) approaches we aimed to characterize the diversity within the Mycobacterium genus and investigate the phylogenetic relationships and functional potential of many poorly known mycobacterial species it is unknown whether the presently accepted phylogeny characterized by several groupings of closely related species remain consistent when considering many NTM species that are still not sequenced but others complexes have been defined and named using a representative species (M The current definition of such groups is mainly based on the presence of genetic signatures at the level of 16S rRNA gene and on shared phenotypic and epidemiologic characteristics we thus aimed at filling the gaps in the current genomic and phylogenetic characterization of the Mycobacterium genus and at providing the basis for the search of new determinants of mycobacterial virulence and antibiotic resistance we sequenced and assembled the genome of 41 new NTM species By analyzing these genomes in the context of the few dozens of already sequenced mycobacterial species we were able to substantially increase our knowledge on the genetic and functional diversity within this genus to provide the foundations of an ultimate phylogeny and to assess the evolutionary impact of horizontal gene transfer Representative species of both rapid and slow growing mycobacteria were included (11 and 36 strains respectively) with preference for those species without available genome in public databases Particular focus was given to the species known to be members of phylogenetically related clades on the basis of 16S rRNA sequences was clearly outside of the phylogenetic branch leading to the latter species and is unquestionably an uncharacterized mycobacterium other than M as was also confirmed by looking at the sequences of most housekeeping genes (data not shown) were phylogenetically very distant and both substantially differing from the type strain of the species (A) The phylogenetic tree (on the left) and the gene presence/absence tree (on the right) are contrasted to highlight the consistency of the complementary evolutionary signals and identifying taxa with potentially uncoupled genetic versus functional evolution (B) The scatter-plot of the pair-wise distance of the strains in the phylogenetic versus gene presence/absence trees (color denotes the average number of ORFs between the compared strains) Both the arrangement of the points and the overall correlation support the consistency between the trees (C) The increase of the pan-genome size as a function of the number of genomes included in the clustering (see Methods) The blue curve highlights the trend for all the genomes (newly sequenced and retrieved from NCBI) whereas the red curve refers to already available genomes only This analysis suggests that genomes sequenced in this work roughly double the gene families available for the Mycobacterium genus as also confirmed by other clustering approaches (see Methods) the high number of hypothetical and broadly characterized proteins in these clusters makes distinguishing their specific functions difficult Nine of these contigs were found to contain either only transposases or transposases plus another gene suggesting the occurrence of transposable elements within the chromosome Three contigs within the same genome were found by homology search to known plasmids (strain 768; M chimaera) and all appeared to be the plasmid pMK12478 derived from M kansasii (86% average nucleotide identity) suggesting a potential horizontal transfer between species belonging to unrelated complexes Several contigs covered different portions of the plasmid suggesting a single plasmid that had not been recovered as a whole during sequencing ranging from 0.11% to 1.81% of genome contents our analysis suggests that it is likely that several circular potential mobile elements portions are present in the genomic repertoire of many Mycobacterium genomes Although many of these appear to likely be phages further sequencing efforts are required to confirm both their identity and function The potential donors of these LGT-derived genes were found to derive from over 130 genera primarily from the Actinobacteria and a small number of Beta-proteobacteria Rhodococcus and Streptomyces) were highlighted as potential donors in over half the species indicating close relationships with organisms belonging to the same class (Actinobacteria) as mycobacteria As members of these genera often share a similar environment (primarily soil-dwelling) it seems likely that most LGT is habitat-driven The large majority of genes acquired through LGT were annotated as genes of unknown function (eggNOG categories R and S and ‘none’, Supplementary Figure 3) The second most frequently transferred category were proteins involved in energy production and conversion Many oxidoreductases were found to be transferred in addition to many other reductases and dehydrogenases Some genomes also acquired carbohydrate and amino acid metabolism and transport genes (categories G and E respectively) and primarily membrane transporters for both These findings suggest that LGT resulted in increased capabilities of growth and nutrient transport Many defense mechanisms were also transferred (category V) allowing for additional rapid protection against antimicrobials perhaps associated with their soil niches or associated with increased capability to infect the human host horizontal acquisition of genes from non-mycobacterial species does not seem to have a large impact on the structure of mycobacterial genomes we found that genes encoding PE/PPE proteins are almost exclusively present in M which is associated to the ESX-3 system and is present in most NTM species The five ESX export systems are heterogeneously distributed across NTM complexes/groups: ESX-3 is conserved in all groups while ESX-1 seems to be specific for M Mycobacterium abscessus group possesses only the ESX-3 system Cell intensity represents the fraction of genes in the corresponding gene family present in a given genome Here we found that the Tat export system is ubiquitous in all the species sequenced in this study SecA2 and YajC proteins belonging to the Sec system are conserved in all the species while the other components of the system are present only in some species but show a significant association with NTM group/complex (p ≤ 0.01) Genes are organized according to their role in MA or DIM biosynthesis pathways; species are grouped according to their assignment to complexes/groups. Only the genes that are absent from at least 2 of the genomes are shown (see Supplementary Table 10 for the complete list) On the left HPLC patterns of mycolic acids for each species are reported Each HPLC pattern is identified by an acronym describing the number and the time of retention of peak clusters in the chromatogram as follows: two continuous sequences of peaks (C2) one early and two late clusters of peaks (E1L2) one early and one late cluster of peaks (E1L1) two early and one late clusters of peaks (E2LI) We applied here whole-genome shotgun sequencing to expand our knowledge on the genomic features of poorly characterized NTMs Our genomic reconstructions of 41 NTM species allowed for an almost doubling of the number of unique gene families occurring within the Mycobacterium genus The analysis revealed an open pan-genome indicating that most of the functional diversity of mycobacteria remains to be characterized our work highlights the diversity of organisms in this genus and provides the first comprehensive whole genome characterization of NTMs Our phylogenetic analysis was based on a genome-wide alignment of hundreds of conserved genes which is recognized as one of the most reliable estimates to study the molecular evolution of organisms On one hand we could confirm previously established relationships of NTM species based on sequencing of a single or few markers In particular we found clearly distinct evolutionary pathways for slow and rapidly growing mycobacteria in agreement with the pre-NGS era phylogeny On the other hand our analysis allowed the reclassification of misplaced species in particular in the M Also the validity of the latter complex was not supported the annotation of the new NTM genomes revealed that a vast majority (>60%) of predicted genes could not be assigned a specific function The validity of most mycobacterial complexes and their functional specialization were highlighted by the high number of genes shared by members of a given complex and absent in the others Although associating known gene functions of group-specific NTM with phenotypic traits is still challenging our sequence-based gene clustering could lay the basis for further analysis aimed to identify markers genes at various taxonomic levels These genes could have potential biomedical applications enabling the development of reliable diagnostic tools for the identification of a large number We selected 47 Mycobacterium species representative of different members of the genus Eleven rapidly growing and 36 slowly growing strains were included Species whose genomes were already present in available databases were not included in our panel With the aim of better understanding the phylogenetic correlations between the species we focused on those that appeared to be members of complexes or groups immunogenum were selected as members of the M Among slow growers the major targets of our investigation were the M avium complex was selected due to the presence of multiple genomes of such group in the databases doricum was included to investigate thoroughly the ambiguous location of this slow grower within the phylogenetic branch of rapid growers; M triviale to make clear its relatedness with the M The other species were selected as representative either of potentially pathogenic or nonpathogenic organisms Strain cultivation was performed using liquid or solid media and incubation at 37 or 30 °C for enhancement of growth For liquid cultures a fully automated system (BACTEC MGIT 960 Mycobacterial Detection System USA) was used while Lowenstein-Jensen and Middlebrook 7H10 agar (BD Diagnostic Systems) were used as solid media rpoB and 16S rRNA genes were partially sequenced by the Sanger method Extracted DNA was quantified using Qubit 2.0 fluorometer (Invitrogen by ThermoFisher Scientific Paired-end libraries were prepared from 1 ng of total bacterial DNA using Nextera XT DNA Sample Preparation kit and Nextera XT Index kit (Illumina Inc. Library concentration and average fragment size were calculated by Qubit 2.0 fluorometer and Caliper LabChip GXI System (Perkin Elmer and sequenced at 10 pM on the Illumina HiSeq 2000 platform (Illumina Inc. USA) with 100 nt paired-end reads to achieve a coverage >100x per base This pipeline consists of three steps: read correction (corrections of sequencing errors based on other reads) read assembly (assembly of the reads and creation of contigs and scaffolds) and mismatch correction (correction of mismatches and short indels in the assembly) No genome was found to have a score lower than 0.82 (average 0.92 ± 0.04) An eggNOG membership is assigned to each protein if 70% of the UBLAST hits belong to the same eggNOG member Distinctions are then made between proteins with no UBLAST hit to any eggNOG sequence (no_hit) hits to a member that is not assigned an eggNOG code (none) and those without a 70% agreement (unassigned) Annotations are also clustered at the 25 higher COG functional category levels as per the eggNOG assignments The statistical tests for the over- or under-representation of genes and gene families in groups of genomes have been performed using the chi-squared test The annotation pipeline (starting from the step of running Prokka) on the raw genomes was performed identically on the new genomes and on the already available ones to avoid potential inconsistencies in the downstream analysis also the number of core genes identified by Roary (179 at 80% protein identity) is consistent In order to detect potential plasmids and (pro)phage sequences within each genome four separate approaches were used: homology read depth and two circular contig detection methods Since only homology-based methods would allow for differentiation of plasmid from phage these two groups were combined in one analysis a contig that was found by any of the below four methods was considered a potential plasmid or phage A hit was retained only if the aligned segments covered at least 90% of the contig and 50% of the plasmid or phage sequence This ensures high identities of partial matches are not retained and increase the likelihood of the genome contig being a whole plasmid or phage this was done using both the sample genome contigs as the query and phage sequences as the database as well as vice versa If the original ends of the contig are joined to form 1 new contig Any such circular contigs over 1 kb in length were retained 500 bp were trimmed from both ends of each contig BLASTn was then used to compare the raw read pairs to these contig ends with an e-value of 1e-10 and a 100% identity If a read has a significant match to one contig end and its pair to the other All contigs found to contain a ribosomal gene were labelled as the chromosome set Any contig found to have an average read depth 10 times greater than the average depth of all contigs in the chromosome set was labelled as a potential plasmid The e-value and percent identity values for HGTector were set as 1e-30 and 80% respectively The core genes concatenated alignment was used as input and any recombination events that were longer than 1 kb were marked as significant The genomes are available 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graphical representation of phylogenetic data and metadata with GraPhlAn Download references Meehan: These authors contributed equally to this work Laboratory of Biomolecular Sequence and Structure Analysis for Health Roberto Bertorelli & Veronica De Sanctis and all authors read and approved the final version of manuscript The authors declare no competing financial interests Reprints and permissions Download citation npj Primary Care Respiratory Medicine (2024) Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research Nibali's team spark a white-knuckle ride down to Tortoli Nibali cagey on his chances at the Giro d'Italia Giro d'Italia: Greipel sprints to stage 2 victory Giro d'Italia: Stage 2 finish line quotes Nibali and his teammates landed a psychological jab to the ribs of Nairo Quintana (Movistar) Geraint Thomas (Team Sky) and anyone else who is hoping to stop Nibali taking a third Corsa Rosa victory Nibali will look for every possible moment to test his rivals during the three weeks of racing The near-20km climb of Genna Silana caused little problem to the peloton only helping to pull back the break of the day Nibali's men in red and gold wanted to stay safe at the head of the pack on the series of long sweeping curves that followed and also tested their rivals' nerves by pushing the speed to a breathtaking limit using every inch of the road and going close to touching the barriers and kerbs The riders behind them had to endure a white-knuckle ride hoping that nobody would let a gap open or slide out on a curve The team confirmed to Cyclingnews that it was a pre-planned strategy decided after a careful recon of the stage and especially the 37 kilometres of sweeping roads down to Tortoli "We decided to lead the race in the last 40 kilometres to avoid risks The first stages are always very nervous and so it's better to stay in the front," directeur sportif Alberto Volpi said following his teammates and copying their trajectories He was also well placed in the sprint to the finish line in another sign of confidence and composure finishing thirteenth behind André Greipel (Lotto Soudal) The first mountain stage to Mount Etna is looming The shark of Messina could be about to bite.  Stephen FarrandSocial Links NavigationHead of NewsStephen is one of the most experienced member of the Cyclingnews team having reported on professional cycling since 1994 He has been Head of News at Cyclingnews since 2022 before which he held the position of European editor since 2012 and previously worked for Reuters