Two hardening environments were used: a carbonation chamber with 5% CO2 and a normal climatic chamber with atmospheric CO2 concentration. The effect of curing times of 1, 3, 7, 14 and 28 days on concrete properties was also analysed. The accelerated carbonation increased the dry bulk density, decreased the accessible porosity water, improved the compressive strength and decreased the setting time to reach a higher mechanical strength. The maximum CO2 capture ratio was achieved with the use of recycled concrete aggregate (52.52 kg/t). Accelerate carbonation conditions led to an increase in carbon capture of 525% compared to curing under atmospheric conditions. Accelerated carbonation of cement-based products containing recycled aggregates from construction and demolition waste is a promising technology for CO2 capture and utilisation and a way to mitigate the effects of climate change, as well as promote the new circular economy paradigm.This research studied the effect of accelerated carbonation in the physical, mechanical and chemical properties of a non-structural vibro-compacted porous concrete made with natural aggregates and two types of recycled aggregates from construction and demolition waste (CDW). Natural aggregates were replaced by recycled aggregates using a volumetric substitution method and the CO During the construction, use, maintenance and subsequent demolition of building and infrastructures, large amounts of construction and demolition waste (CDW) are produced [Since the mid-19th century, the rise of the economy and industry has grown exponentially. This has led to an increase in pollution and greenhouse gas (GHG) emissions, and soon the scarcity of raw materials and natural resources will be more evident [produced ,4,5,6,7 2) is the main greenhouse gas and is one of the main causes of global warming [2. In the construction sector, the manufacture of building materials generates approximately 73% of carbon, of which the manufacture of Portland cement accounts for 41%, hence the scientific and social alarm around the carbon footprint of building materials and their influence on climate change [2 equivalent are produced per cubic metre of concrete [2 irreversibly through the process of cement carbonation [Carbon dioxide 2 or CSH), resulting in calcium carbonate CaCO3 and silica gel, as shown in Equations (1) and (2) [2 can also react with calcium aluminate hydrate to form CaCO3, according to Equation (3) [3S), also called alite, or dicalcium silicate (C2S), also called belite, can further react with CO2, as shown in Equations (4) and (5), respectively [Concrete carbonation is a natural process affected by natural exposure conditions. The progress of carbonation depends mainly on the permeability of concrete, the relative humidity (RH) content and the concentration of CO and (2) ,19,20,21tion (3) ,19,20,21ectively ,21,22,232 concentration, so in order to achieve accelerated carbonation, special curing conditions with a high concentration of CO2 must be used [2 absorbed increases, but the greater the amount of cement per cubic metre of concrete, the lower the CO2 penetration [2 curing makes the microstructure denser, decreases the necessary curing time, decreases permeability and improves mechanical properties [The carbonation phenomenon is based on atmospheric CO be used ,16,24. O be used . As the etration ,25,26. Ioperties ,28.9 tonnes per year [The use of recycled aggregates from CDW in cement-based materials is a viable alternative to reducing the environmental and economic impact in the construction sector. Although it is difficult to measure the current world production of CDW, it is estimated that it exceeds 3.1 × 10per year . Therefoper year . Recycleper year . The preper year ,32 and tper year . Howeverper year ,34,35,362 treatment to improve the properties of cement-based materials made with RA: (i) pre-treatment of recycled aggregates with CO2 and (ii) accelerated carbonation of fresh cement-based mixtures. Although the carbonation mechanisms in both situations are similar, some difficulties arise in the latter. In the case of RA, pre-treatment with CO2 increases the density and decreases the crushing value, water absorption and porosity. It also improves the interfacial transition zone between the carbonated RA and the cement paste, improving the mechanical properties of concrete made with pre-treated RA [There are two possibilities for using COeated RA ,41,42,432 penetrating through the pores of the concrete matrix, which, as it becomes denser, hinders the entry of CO2 and the carbonation of the entire mass. Hence, the porous mixtures, where the entire mass is accessible to CO2, are more suitable for applying new accelerated carbonation technologies [For concrete or mortar made with RA, accelerated carbonation reduces the necessary curing times to reach a certain mechanical strength, water absorption and shrinkage, as well as increasing density and improving mechanical properties. The main problem with accelerated carbonation is that it depends on COnologies ,41,42,43Moreover, the use of porous concrete in pavements allows the infiltration of rainwater, reduces the runoff flow and the damage caused by floods, as well as favours the recharge of aquifers and the growth of trees in cities. Because of this, porous pavements are a very good alternative to mitigate the effects of climate change.This article studies the effect of accelerated carbonation on the physical–mechanical and chemical properties of vibro-compacted porous concrete for the manufacture of eco-friendly precast elements made with the main types of recycled aggregates from CDW (RCA and MRA). It can be divided into three sections: characterisation of the starting materials, the study of the dry bulk density, water absorption, accessible porosity for water and compressive strength of hardened concrete and the carbon capture of the different mixtures and curing methods.Due to the great porosity of this vibro-compacted porous concrete, this study has an added advantage, which is that of a draining property of the runoff from rainfall. In recent years, we have seen more torrential rains due to the effects of climate change, so it would help the evacuation of water more quickly and in an environmentally friendly manner, as we could contribute to the recharge of underground aquifers under our roads made with this porous material.2 capture studies with non-structural porous vibro-compacted concrete made with recycled aggregates from construction and demolition at 28 days of curing, measuring the amount of CO2 sequestered by the concrete using the DTA/TGA technique.There are currently no CO3 was also used.Three types of natural aggregates were used: (i) natural aggregate 0/3 (NA-0/3), (ii) natural gravel 5/7 mm (GN-5/7) and (iii) natural gravel 4/12.5 (GN-4/12.5). Two types of recycled aggregates from CDW obtained from a recycling plant located in Cordoba (Spain) were used: (i) recycled concrete aggregate 2/12.5 (R1) and (ii) mixed recycled aggregate 2/12.5 (R2). The cement used was CEM II/A-L 42.5 R (UNE-EN 197-1: 2011 ). A supe3 according to the data provided by the manufacturer.3, used in each of the mixtures, 3 of the aforementioned aggregates and Three mixtures of porous concrete (PC) were tested: (i) reference, where only natural aggregates were used (PC-REF); (ii) total replacement of GN-5/7 and GN-4/12.5 for recycled aggregate R1 (PC-R1), and (iii) total replacement of GN-5/7 and GN-4/12.5 for recycled aggregate R2 (PC-R2). A volumetric substitution was made and the replacement was carried out according to Equations (6) and (7). The mixtures were made by pouring dry materials from largest to smallest particle size. Later, the saturation water of the aggregates was added and was left for 10 min so that the greatest amount of water possible was absorbed, then the cement and the effective water with the additive were poured. Moulds of 10 cm × 10 cm × 10 cm were used and the concrete mixture was placed in two layers, which were vibro-compacted with a duration of 5 s each layer. The time was adjusted experimentally in the laboratory so that the reference mixture (PC-Ref) had the same density and resistance as the mixture on an industrial scale in the manufacture of draining blocks. This allowed the samples to be demoulded immediately after compaction, just as it is done on an industrial scale.-2 was 0.04% (approximately atmospheric conditions).Normal climatic chamber (NCC): temperature of 21 ± 2 °C and relative humidity of 65 ± 10%. The concentration of CO-2 was different: 5% CO2.Accelerated carbonation chamber (ACC): same temperature and relative humidity as that for NCC. Only the concentration of COTwo types of hardening environments were used:−1. In hardened concrete samples, the XRD pattern was analysed at 28 days. The hardened samples were ground to a powder to obtain a representative sample. The samples were immersed in ethanol for the desired curing age for 48 h, in order to stop the hydration reactions of the cement at that age of study [Raw materials (aggregates and cement) and hardened concrete samples were characterised by X-ray diffraction (XRD). Bruker D8 Discover A25 equipment with CuKα  was used. Diffraction patterns were measured between 10° and 70° (2θ) at a rate of 0.006 θ minof study ,46,47.To determine the elemental chemical composition of the raw materials, a wavelength dispersive X-ray fluorescence spectrometry (XRF) analysis was carried out with a power of 4 kW and a ZSX PRIMUS IV (Rigaku).−1. The test temperature ranged from room temperature to approximately 1000 °C. The TGA indicates the mass at each temperature and the DTA indicates the differential thermal analysis. Differential thermal analysis (DTA) measures endothermic and exothermic transitions as a function of temperature. This is useful for understanding the temperature range in which significant phase changes occur for inorganic compounds. With TGA, we can observe the variability of mass as a function of temperature.Thermogravimetric analysis (TGA) and differential thermal analysis (DTA) were performed for raw materials and hardened concrete samples at 28 days of curing. TGA was performed on a Setaram Setsys Evolution 16/18 apparatus at a heating rate of 5° minThe carbonation depth was determined by spraying a freshly divided surface with phenolphthalein  according to the standard UNE-EN 14630:2007 . The carThe compressive strength (CS) was measured in accordance with the European standard UNE-EN 12390-3:2020  at the cThe determination of the water absorption, dry bulk density and water-accessible porosity of the concrete was carried out in accordance with the European standard UNE 83980:2014 .A particle size distribution analysis was carried out according to the European standard UNE-EN 933-1:2012 . The fir2 and CaO, although it should be noted that the amount of SiO2 is significantly higher than that of CaO, which may be due to the nature of the sands or coarse sand used for the manufacture of mortar or concrete from which this waste comes. These values are consistent with those obtained by Suescum et al. [2 and Al2O3 with respect to R1. This is due to the incorporation of the ceramic particles that make up this aggregate, given that the manufacture of the ceramic material contains a large amount of silica (SiO2) and alumina (Al2O3) [m et al. . It can  (Al2O3) .3) (05-0603) [2) (05-0490) [3) (05-0586) [3Al)O8) (10-0393) [2AlSi3O10(OH)2·H2O (15-0603) [2SiO4) (09-0351), portlandite (44-1481) and gypsum (21-0816) [3O8) (89-8574) [3S)(86-0402), brownmillerite (11-0124) [The X-ray diffraction patterns of the raw materials used for this study are shown in 05-0603) . It was 05-0603) . For agg05-0490) , while o05-0586) , and oth10-0393) , illite 15-0603) , belite(21-0816) . In addi89-8574)  is also 89-8574) . The pha11-0124) , calcite11-0124) , gypsum 11-0124)  and port11-0124) , which itlandite -1481 and11-0124) ,56,57,58For aggregates from CDW (R1 and R2), three sections were identified: (i) the loss of moisture that can be seen from room temperature to 105 °C; (ii) the loss of aluminates and calcium silicates from 105 °C to 430 °C; (iii) and the last section, which is the decomposition of calcium carbonate (according to Equation (8)) that ranges from 430 °C to 1000 °C. For R2, a peak at 120 °C (between 105 °C and 130 °C) was observed, which is due to the decomposition of gypsum, identified by XRD . In addiIn TGA/DTA of cement, four stages can be observed: (i) from room temperature to 105 °C, which corresponds to the elimination of moisture; (ii) from 105 °C to 380 °C, dehydration of aluminates and calcium silicates takes place ,53; , it was observed that the dry bulk density values increased and that the water absorption and accessible porosity for water increased. The lowest dry bulk density obtained is for the PC-R2 mix, which also leads to the highest porosity. These results are in accordance with previous studies on the use of recycled masonry aggregates for the manufacture of concrete blocks for masonry mortars [2 (see 3) during the carbonation process, shown in Equations (1)–(5). The appearance of this compound fills the pores of the hardened sample, increasing the dry density and decreasing the porosity [Regarding the effect of curing in a CO mortars . This iss [2 see , caused porosity ,38,40.In the NCC curing environment, mixes made with RA (PC-R1 and PC-R2) at all curing ages showed lower compressive strength mean values than the reference mixture (PC-REF), even though the substitution was made using the volumetric method that usually gives better results than substitution by weight ,76,77,78In the case of the ACC environment, the average value of the compressive strength of PC-R1 was reduced by an average of 22.6% (range 10–36%), although in this case, the curing time significantly affected this percentage of reduction. The difference was higher at early stages and lower after 14 days of curing. On the contrary, the PC-R2 mixture showed a reduction in compressive strength of 18% compared to PC-REF with one day of curing, but its mechanical strength evolved very favourably over time, with PC-R2 showing compressive strength mean values slightly higher than PC-REF at 3, 7 and 14 days of curing and similar values at 28 days.2 curing.The mean compressive strength values of the PC-REF mixtures significantly increased under accelerated carbonation conditions. The mechanical strength increased by more than 36% at one day of curing, which can be beneficial for the precast industry since they can reduce the curing time products and productivity of the factory, lower energy consumption, or even reduce the amount of cement and carbon footprint of the precast element . In the 2 curing environment could be due to the carbonation of the mixture and thus to the slight densification of the mixture, as can be seen in For the mixture made with R2 aggregate (PC-R2), the effect of carbonation appeared more prominent at 3, 7 and 14 days, even exceeding the compressive strength mean values of the mixtures made with NA (PC-REF). Similar conclusions were found by Suescum et al. in other studies carried out using mortars made with mixed recycled aggregates ,22,26. T2 environment was more effective when using this type of aggregate, which can be explained by the fact that the particles of aggregates R1 and R2 are more porous than NA –(5)). However, the percentage of growth in mechanical strength decreases at later stages, as can be observed for PC-R2/ACC, where the increase between day 14 and day 28 was less than 3%. This can be attributed to the calcium carbonate formed during the first days of curing with high concentrations of CO2 densifying the pores and preventing the entry of CO2 and the consequent carbonation reactions in the internal concrete matrix. Similar conclusions were drawn by Pingping et al. [Finally, the highest increases in the COg et al. .For the PC-REF/28DAYS/NCC mix, one main phase was identified as calcite (05-0586) , which iHowever, for the PC-REF/28DAYS/ACC mixture, a majority phase was identified as calcite (05-0586)  and a veFor the samples made with R1 (PC-R1), the same main phases were identified in both ACC and NCC, quartz (05-0490) , calciteFor the samples with R2 aggregate (PC-R2), the same main phases were identified in both ACC and NCC as occurred with the mixture made with R1 aggregate: quartz (05-0490) , calciteIt is observed that for samples PC-REF/28DAYS/NCC and PC-R1/28DAYS/NCC, there was an average carbonation depth of 3 and 4 mm, respectively, while for sample PC-R2/28DAYS/NCC, no carbonated part was observed.Samples PC-REF/28DAYS/ACC, PC-R1/28DAYS/ACC and PC-R2/28DAYS/ACC had an average carbonation depth of 23.5, 34.6 and 22.5 mm respectively.2 sequestration and therefore an increase in mass from the densification of the aggregate and therefore of the mixture [Therefore, it can be said that the substitution of NA for RCA favours carbonation, due to the increased porosity of the RCA aggregate as opposed to the lower porosity of NA, which contributes to greater CO mixture ,61,80.It can also be observed that in the mixtures PC-REF/28DAYS/ACC and PC-R2/28DAYS/ACC, the carbonation fronts are irregular due to the irregularity of the interconnected pores, so that in addition to this qualitative test, it is convenient to carry out TGA/DTA analyses and to account for the carbonated part in a more exhaustive way.(i)From room temperature to 105 °C. In this phase, the loss of physically absorbed water was observed, i.e., moisture was lost. The mixtures PC-R1 and PC-R2 showed a higher moisture content related to the higher water absorption presented by R1 and R2, observable on practically all the curing days, since the DTA peak is higher than for the reference simples [ simples ,47.(ii)In the second stage, from 105 °C to 400 °C, dehydration of the calcium silicates and aluminates took place, also according to the analyses carried out on the raw materials previously used [sly used .(iii)In the third stage, from about 400 to 480 °C, the decomposition of portlandite occurred, which was identified by an endothermic peak at around 410–420 °C. For the case of the reference mixture, a peak was found for all curing times in the normal climatic environment. However, for this same mixture in the CO2 curing environment the portlandite disappeared for all the curing times studied. This indicates that the carbonation is complete as indicated by XRD. This is also in accordance with the observed improvements in compressive strength (see ngth see . For the(iv)The fourth stage, ranging from 480 to 640 °C, comprised the section where weight loss occurred due to the initial decomposition of carbonates formed during the hardening process [ process .(v)in the fifth and last stage from 640 to 1000 °C, decomposition of calcium carbonate occurred [Finally, occurred .A five-stage division was observed:2 sequestered (as a percentage) by the different samples, curing environments and days of curing were calculated from the TGA/DTA analysis. A summary of the results can be seen in 2 absorption according to Equation (9) This is because when comparing two equal samples, in which the only variant from one chamber to the other is the CO2 concentration, it is obvious that the difference corresponds to the sequestered CO2, shown as a product of the carbonation reaction.Once all the TGA/DTA analyses of the hardened samples for both curing environments were shown, the amount of CO3, which is shown in Mass losses between 105 °C and 400 °C were assigned to the setting water of the cement, which is the water of the hydrated calcium silicates and aluminates. Losses between 400 °C and 480 °C were attributed to portlandite. Finally, the fourth and fifth TGA/DTA stages were unified to quantify the decomposition of CaCO2 in the environment can be considered almost zero. For the PC-REF sample, the amount absorbed was 1.6 kg CO2/t (0.1592%) at curing of 7 days. The maximum capture capacity of the PC-REF sample was at 28 days, with a value of 12.37 kg/t CO2. The large difference in this sample between the curing for 7 and 28 days is noteworthy, with an increase of 700%, with this material serving as a CO2 sink.The amount absorbed by the samples cured in the NCC environment has been considered a reference, although in reality a small sequestration would occur, which for practical purposes we consider negligible because the amount of COFor all curing ages, the PC-R1 mixture had the highest absorption. This absorption increased in turn with the age of curing, obtaining at 28 days an absorption of 52.52 kg/t, the maximum value recorded among all the mixtures and duration of curing. For this mixture, there is an increase of 30% when compared to the 7-day stage, where absorption value was 40.48 kg/t. Compared to the mixture PC-REF, it obtained between 240% and 333% more absorption, depending on the curing stage.2 capture properties than the concrete waste due to the cement paste adhered to it.The PC-R2 sample is the one that obtained the worst data, reaching 5.5 kg/t after 28 days of curing, lower than the value obtained by the other two mixtures, leading to the conclusion that the ceramic material has worse CO2 sequestration capacity compared to the normal chamber by 123%, 525%, and 55% for the PC-REF, PC-R1 and PC-R2 mixtures, respectively.Accelerated carbonation increased the CO2 absorption, obtaining values of 52.52 kg/t, which is an increase of 525% with respect to curing in an NCC environment. If we add to this the use of RCA, we also obtain improvements of 333% with respect to the mixture composed of natural aggregates. These results reveal that accelerated carbonation of cement-based products containing recycled aggregates is a promising technology for CO2 capture and utilisation, making CO2 a potential industrial feedstock.It is therefore clear that curing in an ACC environment means a great increase in CO2 capture of a porous vibro-compacted concrete for use in precast products that incorporate two types of recycled aggregates from construction and demolition waste, yielded the following conclusions:2 sequestration advantage.The substitution of recycled concrete aggregate (R1) presented a very significant COAccelerated carbonation reduced the curing time (setting of the mixture in less curing time), reduced water accessible porosity, and increased the dry bulk density compressive strength mean values, especially during early stages of curing (1–14 days).2.In vibro-compacted concrete samples subjected to accelerated carbonation, a decrease or disappearance of the portlandite phase and an increase in calcium carbonate was observed using TGA/DTA and XRD techniques, with the consequent sequestration of CO2 sequestration at 28 days for the PC-REF , PC-R1 (recycled concrete aggregates) and PC-R2 (mixed recycled aggregates) mixtures was approximately 12.36 kg/t, 52.52 kg/t and 5.5 kg/t, respectively, representing an increase of 123%, 525%, and 55% over the curing under an atmospheric environment.The amount of COThis study on the effect of accelerated carbonation on the physical–-mechanical and CO2 capture and utilisation in precast products. The main advantages for the precast concrete industry would be, among others, the significant improvement in the mechanical performance of the cement-based materials, reduction in curing times, reduction in the amount of cement to be used to achieve similar mechanical strength in CCN curing conditions, and a lower carbon footprint per cubic metre of concrete. In addition to this, the use of recycled aggregates from construction and demolition waste would promote the new circular economy paradigm and the CO2 sink effect of precast concrete elements, which could mitigate climate change.This study indicates that accelerated carbonation of porous vibro-compacted concrete containing recycled concrete aggregates and mixed recycled aggregates is a promising technology for CO


The National Action Plan on Antimicrobial Resistance in Bhutan promotes the rational use of antibiotics. It is important to establish baseline data on the use of antibiotics and the quality indicators of antibiotic prescriptions to identify where improvement efforts may need to be focused.To describe the prevalence and patterns of antibiotic prescription and establish baseline data regarding quality indicators of antibiotic prescriptions in four major hospitals in Bhutan.This was a point prevalence survey of antibiotic use among inpatients in June 2022 conducted using the Australian National Antibiotic Prescribing Survey (NAPS).There were 314 patients (41.5%) receiving at least one antibiotic on the audit day. Among prescriptions reviewed, 278 (88.5%) had indications for use documented, 102 (32.5%) had a review or stop date documented and 120 (38.2%) had microbiology samples collected prior to antibiotics. Ceftriaxone , cefazolin  and metronidazole , were the common antibiotics prescribed. The most common indications for use were surgical prophylaxis , community-acquired pneumonia  and sepsis . There were 125 prescriptions (39.8%) that were compliant with national/therapeutic antibiotic guidelines and 169 (53.8%) where antibiotic prescriptions were appropriate.This study identified key areas for targeted interventions in antimicrobial stewardship programmes in Bhutan. The prevalence of antibiotic use, indications for use, and drug choices were similar to data from other countries. Documentation plans for durations of use, prolonged surgical prophylaxis and concordance of choices with guideline recommendations present opportunities for improvement. One common factor that is attributed to rising rates of AMR is the inappropriate and excessive use of antibiotics.3 Antibiotic use and antibiotic prescription are influenced by many factors that can collectively be addressed through antimicrobial stewardship initiatives.5Antimicrobial resistance (AMR) has become a major threat to global health and was associated with an estimated 4.95 million deaths in 2019.7 The National Antimicrobial Prescribing Survey (NAPS) is a voluntary web-based qualitative auditing platform that has been adopted by over 75% of all public and private facilities in Australia since its release in 2013 and successfully used in Canada, New Zealand, the UK, Fiji, Malaysia, Papua New Guinea, Timor-Leste and Vietnam.8Measuring, monitoring and evaluating antibiotic prescribing practices in hospitals is important to ensure that context-specific stewardship initiatives are developed and implemented effectively. There are several available tools to do this, including point prevalence survey protocols from the WHO.9 National antimicrobial prescribing guidelines were developed and endorsed in 2018 and implemented across all hospitals.10Bhutan is a country with a population of 0.7 million situated in the eastern Himalayas. It has universal access to free public healthcare, and health policies are centrally regulated by the Ministry of Health. The country has demonstrated a strong commitment to AMR-related policies and activities. Bhutan endorsed the National Action Plan on Antimicrobial Resistance in 2017 with a One Health focus.Antimicrobial stewardship programmes were established at the Jigme Dorji Wangchuck (JDW) National Referral Hospital, Thimphu in 2016 and at the Central Regional Referral Hospital in Gelephu, Eastern Regional Referral Hospital in Monggar and Phuentsholing General Hospital in 2019. These four hospitals were designated as sentinel sites for the surveillance of antimicrobial use under the Fleming Fund project. The Fleming Fund is a UK Aid programme that supports low- and middle-income countries to generate, share and use data to improve antimicrobial use and encourage investments in AMR. The antimicrobial stewardship programme at the JDW National Referral Hospital is delivered by a team that includes a clinical microbiologist, a pharmacologist, physicians and nurses. The antimicrobial stewardship programmes in the other hospitals are delivered by physicians, nurses and pharmacists. Some of the antimicrobial stewardship interventions in these hospitals include education and training of staff, an approval system to manage access to restricted broad-spectrum antibiotics, surveillance of antimicrobial consumption and dedicated audits of specific antibiotic use.This study was conducted hospital-wide to obtain baseline information on antibiotic prevalence, the most common antibiotics prescribed, the appropriateness of use as per national guidelines and quality indicators of antibiotic prescriptions in the four major hospitals in Bhutan. The aim was to identify areas for focused interventions to optimize the use of antibiotics in hospitals and contribute to efforts towards preventing and reducing AMR in Bhutan.This was a point prevalence survey of antibiotic use in four major hospitals in Bhutan: JDW National Referral Hospital (380 beds) in Thimphu, Central Regional Referral Hospital (150 beds) in Gelephu, Eastern Regional Referral Hospital (150 beds) in Monggar and Phuentsholing General Hospital (60 beds).11 The JDW National Referral Hospital, Central Regional Referral Hospital and Eastern Regional Referral Hospital provide tertiary-level care and serve as strategic referral points in their region. They are equipped with specialist services such as ICUs, haemodialysis units and surgical services. These three hospitals are designated as teaching hospitals with intern medical doctors and postgraduate trainees among the prescribers. Phuentsholing General Hospital is a secondary-level hospital but is equipped with blood culture, haemodialysis and surgical services as it caters to a relatively larger population. The JDW National Referral Hospital caters to the largest patient load, followed by the Central Regional Referral Hospital. The number of doctors and specialists posted is highest at the JDW National Referral Hospital. While patients are expected to follow a referral pathway of accessing care at the primary-level hospital followed by the designated district/general hospital, it is often a common finding that patients jump the referral pathway and present directly at the tertiary-level hospitals.Bhutan has a three-tiered healthcare system with 10-bed hospitals at the primary level, district and general hospitals at the secondary level and three referral hospitals at the tertiary level.The data were collected between 2 and 18 June 2022. All inpatients admitted at 8 am on the day of the audit were eligible. The patients admitted for day-care procedures were excluded. The patients’ medical records were reviewed to capture patients on current antibiotics and single-dose antibiotics administered within the previous 24 h.8 It is an online auditing platform with a standardized point prevalence survey suitable for use in all hospitals to support real-time data collection and reporting with dashboards and aggregation of data for benchmarking. Prescriptions of topical antimicrobials, systemic antifungals, systemic antivirals and antimycobacterial agents were not included in auditing for this survey.This study adopted the NAPS tool that was developed by the National Centre for Antimicrobial Stewardship, Australia, in 2013.6 antimicrobial prescription is initially recorded as follows: ‘optimal’ if prescription follows therapeutic guidelines or has been reviewed by an infectious diseases clinician or clinical microbiologist; ‘adequate’ if the prescription does not optimally follow therapeutic guidelines but a reasonable alternative choice of antimicrobial is selected; ‘suboptimal’ if the choice of antibiotic is unreasonable or there is an allergy mismatch; ‘inadequate’ if the choice is unlikely to treat the likely causative agent; or it may be ‘not assessable’ if information such as indication is not documented. Those prescriptions recorded as ‘optimal’ or ‘adequate’ are categorized as ‘appropriate’ and those that are recorded as ‘suboptimal’ or ‘inadequate’ are categorized as ‘inappropriate’. In our study, the National Antibiotic Guideline10 was taken as the standard therapeutic guideline for the country.The survey was conducted by 12 local auditors in Bhutan . The auditors were trained on the NAPS data collection protocol and on the assessment of ‘compliance with guidelines’ and ‘appropriateness’ by Australia-based staff. According to the NAPS protocol,When there were any difficulties in performing assessments during data collection, clarifications and support were provided by the focal person for the antimicrobial stewardship programme at the JDW National Referral Hospital and difficulties were further discussed with the NAPS staff in Australia.6 The data were extracted into Microsoft Excel and the variables of interest were analysed. Categorical data are presented as frequencies and percentages and continuous variables are summarized as mean and standard deviation.The data collected were de-identified and entered in the NAPS online database by the auditors.This study was conducted according to the principles of the Declaration of Helsinki. Administrative approval was obtained from the Policy and Planning Division, Ministry of Health Bhutan, and the hospital administrators of the participating sites.There were 756 patients captured during the study period: 487 at the JDW National Referral Hospital, 78 at the Central Regional Referral Hospital, 112 at the Eastern Regional Referral Hospital and 79 at Phuentsholing Hospital. There were 314 patients (41.5%) patients receiving at least one antibiotic on the audit day: 206 (42.3%) at JDW National Referral Hospital, 42 (53.8%) at Central Regional Referral Hospital, 38 (33.9%) at Eastern Regional Referral Hospital and 28 (35.4%) at Phuentsholing General Hospital. The basic description of the patients and antibiotic use in the four hospitals is shown in Table Among the prescriptions audited, the indication for use was documented in the prescriptions of 278 patients (88.5%) and a review or stop date was recorded in 102 (32.5%). The common indications for which antibiotics were prescribed included surgical prophylaxis , community-acquired pneumonia  and sepsis . Among patients receiving antibiotics for surgical prophylaxis, 22 (52.4%) had received antibiotics longer than 24 h.12 are shown in Figure Across all hospitals, ceftriaxone 68; 21.7%), cefazolin  and metronidazole  were the most common antibiotics prescribed. There were 190 (60.5%) patients on one antibiotic, 112 (35.7%) on two antibiotics and 12 (3.8%) on three antibiotics. The top 10 commonly prescribed antibiotics and the WHO Access, Watch and Reserve (AWaRe) groups.7%, cefa10 and there was no appropriate guideline available for 15 (4.8%) cases. There were only 26 patients (8.3%) in whom the prescription had changed from empirical to directed therapy based on microbiology findings. Overall, there were 169 patients (53.8%) who were receiving antibiotics that were inappropriate. The reasons for inappropriate prescriptions were the spectrum of antibiotic prescribed being too broad for the indication , incorrect duration , incorrect dose or frequency , antibiotic not indicated , spectrum too narrow  and incorrect route . The indications for antibiotics and the appropriateness of prescriptions are shown in Figure There were 134 prescriptions (42.7%) that were non-compliant with the National Antibiotic Guideline13–15 While studies from other settings report the highest prevalence of antibiotic prescription in ICUs, our data report the highest volume of patients on antibiotics in Internal Medicine wards.To our knowledge, this is the first survey in Bhutan that describes the prescription and use of antibiotics among patients admitted to four major hospitals that cater to large patient loads. The overall antibiotic prescription rate was 41.5%, with the highest proportion reported at the Central Regional Referral Hospital. The prevalence of antibiotic prescription is within the range of 37.3%–49.0% among inpatients reported in samples from within the region including India, Nepal and Malaysia.13–15 It is important to note that Bhutan does not have a medical college and all its doctors/prescribers are trained in the countries in the region. Ceftriaxone is available only at secondary- and tertiary-level centres in Bhutan as per the National Essential Medicines List 2021.16 The overuse of ceftriaxone is a growing concern, with reports of increasing resistance to β-lactams and cephalosporins. Resistance to ampicillin (2.1%) and ceftriaxone (1.5%) was most the common in bacteriologically confirmed blood stream infections with Escherichia coli, according to the WHO Global Antimicrobial Resistance and Use Surveillance System (GLASS) report 2020 from Bhutan.17In all hospitals, ceftriaxone was the most common antibiotic prescribed, followed by cefazolin, metronidazole and ampicillin. Ceftriaxone was one of the top antibiotics prescribed in studies reported from Nepal (32.7%), India (21.5%) and Malaysia (9.5%).10 varied across hospitals. The majority of the prescriptions at the Central Regional Referral Hospital and Eastern Regional Referral Hospital were concordant with the national guidelines while JDW National Referral Hospital had much higher non-concordance observed. The JDW National Referral Hospital has a higher number of prescribers and trainee doctors, and receives many cases in critical conditions. The two regional referral hospitals have fewer number of prescribers and may also have a less complex patient case mix; however, further exploration on compliance to antibiotic guidelines is warranted.The top indication for antibiotics was surgical prophylaxis, and the majority of these patients received antibiotics for durations longer than 24 h. This is a key area for attention as rates seem much higher than in other countries. The appropriateness of antibiotic choice and compliance with the National Antibiotic Guideline18 there are no established means to assess, audit and enforce these regulations on day-to-day practices. The Bhutan Food and Drug Administration has an established reporting mechanism for adverse drug reactions; however, history taking and documentation of drug allergy was inadequate for evaluation using the NAPS. With the Digital Drukyul project that will implement an electronic patient information system (ePIS), some of these concerns are expected to be addressed. It is recommended that there is a need for regular training of prescribers to adopt good prescribing practices.There were several instances of poor documentation noted. In Bhutan, all medical records are on paper-based files that have open-ended space for prescribers to document clinical details. While good prescribing practices are recommended as per the standards of the Medical and Health Professionals Council and the Bhutan Medicines Rules and Regulations 2019,This survey also identified the lack of collection of appropriate microbiology samples prior to the commencement of antibiotics. These four hospitals are equipped with microscopy and culture services that received upgraded facilities through the Fleming Fund project. The findings from this survey suggest a high proportion of missed opportunities for microbiology sampling that would have potentially guided more appropriate antibiotic therapy. Overall, microbiology-guided therapy was very low across all hospitals. This finding might have been due to this survey being a single point prevalence snapshot so had failed to follow through with how prescriptions are revised based on microbiology reports. However, it is recommended to provide continuing medical education to prescribers on the adoption and utilization of microbiology testing with a target of reducing inappropriate antibiotic prescriptions.9 Auditing and feedback about antimicrobial prescribing quality, with the use of this standardized and validated point prevalence survey tool, is now intended to be conducted at regular intervals to monitor improvement.The adoption of the Australian NAPS tool was effective and user-friendly and there are plans to roll it out to other district hospitals. One of the advantages of this tool is the data analysis dashboard that helped hospitals establish baseline data and plan their target interventions. The findings from this study have identified the areas of interventions that can help promote stewardship initiatives to help meet the targets of the National Action Plan on Antimicrobial Resistance in Bhutan.Activities for the prevention of AMR are now adopted at all levels of hospitals. The four hospitals have developed an annual workplan with specific activities under six areas of interventions: building leadership commitment; establishing accountability and responsibility of delivering the antimicrobial stewardship programme; key activities such as implementation of guidelines, conducting audits and establishing surveillance systems for hospital-acquired infections; education and training activities; monitoring and evaluation of activities; and reporting and feedback within the health facilities. The overall activities are a part of the hospital’s key performance activities and are reported to the national antimicrobial stewardship programme. The summary dashboard data generated from the NAPS have been given to the respective hospitals as their baseline data for targeted intervention within their facilities.The overall antibiotic prescription prevalence among inpatients in the four major hospitals in Bhutan is comparable to that from hospitals in the region. Opportunities to improve documentation, encourage use of the national antibiotic prescribing guidelines, and address surgical prophylaxis duration were highlighted. The Australian NAPS tool was successfully adopted in Bhutan and is planned to be used in other hospitals to support antimicrobial stewardship.


Campylobacters’ ability to enter a viable but non-culturable (VBNC) state, remaining “viable” but unable to grow on culture media. We explored the persistence of VBNC states in specific environments, particularly at broiler farms, as this state may lead to an underestimation of the present Campylobacter prevalence. For VBNC detection, a propidium monoazide PMA-dye viability qPCR (v-qPCR) was used in combination with cultivation methods. We examined samples collected from broiler farm barns and their surroundings, as well as chicken manure from experimental pens. In addition, the tenacity of culturable and VBNC-Campylobacter was studied in vitro in soil and water. In a total of three visits, Campylobacter was not detected either culturally or by v-qPCR (no Campylobacter DNA) in the environment of the broiler farms. In four visits, however, VBNC-Campylobacter were detected both inside and outside the barns. The overall prevalence in environmental samples was 15.9% for VBNC-Campylobacter, 62.2% for Campylobacter DNA, and 1.2% for culturable C. jejuni. In the experimental pens, no cultivable C. jejuni was detected in chicken manure after 24 h. Strikingly, “VBNC-Campylobacter” persisted even after 72 h. “VBNC-Campylobacter” were confirmed in barn surroundings and naturally contaminated chicken manure. Laboratory studies revealed that VBNC-Campylobacter can remain intact in soil for up to 28 days and in water for at least 63 days, depending on environmental conditions.Campylobacteriosis cases in humans are of global concern, with high prevalence rates in the poultry reservoir considered the most important source of infection. Research findings show  Campylobacter infections, with 127,840 recorded cases in the EU in 2021 [Campylobacter enters the food chain. Most cases (20–30%) of human campylobacteriosis in the EU are attributed to the consumption of poultry meat. A significant proportion (50–80%) is thereby causally linked to the high prevalence of Campylobacter (C) C. jejuni and C. coli in the poultry reservoir. Campylobacter occurrence in poultry production is currently associated with emerging antimicrobial resistances to antibiotics. [Campylobacter manages to colonize new flocks despite proper interventions remains incomplete. Some studies have provided valuable insights into the epidemiological situation using cultivation and molecular epidemiology. Notably, the environment is frequently mentioned as a reservoir for Campylobacter at chicken farms. However, the results of different studies suggest that cultivating Campylobacter in broiler farm environments remains challenging [Campylobacter when exposed to various environmental conditions. Exposure to various environmental stressors, including oxidative stress, starvation, osmotic stress, temperature, pH, and UV light, has been discussed to induce a viable but non-culturable (VBNC) state in Campylobacter [Campylobacter was first described by Rollins and Colwell for its survival in natural aquatic environments [Campylobacter in the environment of broiler farms and subsequent colonization of broilers may also be related to the VBNC state. Previous studies have demonstrated that VBNC-Campylobacter (C. jejuni) can resuscitate in vivo or under laboratory conditions, as well as express pathogenicity [Campylobacter in food processing conditions. [Campylobacter, polymerase chain reaction (PCR) methods are used; however, they only amplify the total DNA (from both viable and dead cells). PMA pre-treatment combined with qPCR has been used in various investigations to confirm viable Campylobacter [Campylobacter in poultry and environmental samples, a pretreatment step involving PMA and ISPC was employed. Subsequently, following the photoactivation of the dye, the samples were analyzed using qPCR. To further expand the understanding of VBNC states in the environment, (i) the tenacity of culturable Campylobacter, (ii) the stability of VBNC Campylobacter induced in raw milk, and (iii) the transition of culturable C. jejuni into the VBNC state in vitro was investigated.Poultry meat is a significant source of cases 20–0% of humoportion 0–80% is biotics. . Despitellenging ,10. Thislobacter ,14,15. Tronments . Subsequronments ,18,19,20genicity ,20,21. Fditions. . As cultlobacter ,24,25. Hlobacter ,27. In tlobacter  was used2 for 36–42 days, while Hubbard broilers (farm B) were stocked at 25 kg/m2 for 60 days. The chickens received a three-phase feeding diet matching the commercial standards. Access to outdoor areas was not provided. Thinning procedure was carried out approximately one week before the entire flocks were removed. The three rural farms are surrounded by fields, forests, and small artificial waterways with adjacent lakes present at distances of 0.5 to 1.5 km. The farms followed several biosecurity measures, including personal hygiene practices, disinfectant footbaths or mats and cleaning and disinfection of the broiler houses as specified in guidelines by the German Agricultural Society (DLG) and the German Veterinary Society (DVG).For the first part of the study , seven visits to broiler farms (A-C) were conducted between November 2019 and September 2020 . All farCampylobacter-contaminated chicken manure (harboring the strain BfR-CA-14430) [The second part of the study  was conducted at the experimental facilities of the Centre for Infection Medicine within the Department of Veterinary Medicine at Freie Universität Berlin. The investigation was carried out after the removal of the flocks. The natural A-14430)  from fouCampylobacter. In the second trial, the stability of VBNC C. jejuni induced in raw milk was observed and determined. In the last part, a combination of both trials and a further determination of the transition of cultivable C. jejuni into their VBNC state was investigated. Soil and water were used as experimental matrices, and all trials were carried out in three different microhabitats, each characterized by unique features as outlined in The third part of the study was a laboratory-based in vitro study , which was split into three different trials. The first trial aimed at investigating the tenacity of cultivable ®-Faltenfilter, Typ 601P Carl Roth, Karlsruhe, Germany) and sterile glass funnels. Filtered samples were centrifuged at 4 °C  for 15 min, the supernatant discarded, and the pellet resuspended in 3 mL PW. The final sample was then divided into aliquots as follows: 1 mL reserved for cultivation and 2 mL for differentiation of live and dead cells using quantitative polymerase chain reaction (v-qPCR). Air samples  were collected as described previously [® µ cones were transferred to a 15 mL sterile screw cap tube , and the sample was then centrifuged at 4 °C  for 15 min. The supernatant was discarded, and the pellet was resuspended in 3 mL PW and aliquoted. From each water sample, 50 mL was either filtered and centrifuged or directly centrifuged, depending on the level of visible pollution.Sampling was conducted at each farm after the broiler thinning procedure was carried out. Environmental samples  were collected outside the barns, as described previously . After sChicken manure was collected at 24 h intervals from 0 to 72 h after flock removal from four separate animal rooms. Per room, chicken manure was divided into four different areas , which were distinguished in terms of moisture and dryness. Approximately 30–50 g of manure was collected with sterile spatulas and transferred into sterile 120 mL specimen containers. Manure samples were then prepared and handled as described above.g for 1 min to eliminate soil particles, sand grains, and other organic matter that could not be readily filtered and may have a detrimental impact on DNA extraction and v-qPCR. Subsequently, the supernatant was transferred to a sterile tube and centrifuged again at 600× g for 1 min. Finally, the supernatant was homogenized and aliquoted as described above.Water from the experimental trial 2 was vortexed, and 3 mL of the homogenized mixture was transferred to a sterile 15 mL tube, gently vortexed again, and aliquoted as described previously. Soil samples were collected using sterile spatulas and placed into sterile screw cap tubes (50 mL) . Afterwards, samples were diluted 1:2 in 3 mL of PW and homogenized by vortexing. For the determination of colony-forming units (CFU) (cultivation), 1 mL of the suspension was used. For PMA-v-qPCR analysis, 2 mL of the sample mixture was centrifuged at 600× C. jejuni strain (BfR-CA-14430), preserved at −80 °C, was cultivated on Columbia agar supplemented with 5% sheep blood  for 24 h at 42 °C under microaerobic conditions  in a tri-gas incubator . After sub-culturing in BHI with twice the amount of Growth Supplement  for 18 ± 2 h, cells were suspended in BHI and adjusted to an optical density of 0.2 at a wavelength of 600 nm (OD600), equivalent to approximately 9 log10 cell counts per ml as reported earlier [10 CFU/g or ml. The VBNC-C. jejuni cells (BfR-CA-14430) in raw milk (~7 log10 viable cells/mL) were provided by the German National Reference Laboratory (NRL) for Campylobacter [10/g or ml.For the laboratory-based study in experimental trial 2, the  earlier . This sulobacter  and spikCampylobacter in experimental trial 1 were subjected to quantitative assessment using enrichment, following the ISO 10272-1:2017 [Campylobacter selective Supplement , Growth Supplement , and defibrinated horse blood , then incubated in a microaerobic atmosphere at 41.5 °C for 24 h. Enriched cultures were inoculated onto selective mCCDA plates using a sterile 10-μL loop, followed by incubation at 41.5 °C under microaerobic conditions for another 48 ± 2 h. Putative colonies were isolated and streaked on Columbia blood agar with 5% sheep blood and then incubated as described above. Colonies were analyzed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry . When Campylobacter growth in experimental trial 2 was not achievable following ISO 10272-2:2017, putative VBNC cell suspensions (100 µL) from 1 mL reserved suspension from pre-treatment were transferred to supplemented PB and incubated under similar conditions for up to 72 h to confirm the absence of viable C. jejuni.Quantification was evaluated via colony forming units (CFU) following ISO 10272-2:2017 . Pre-tre2-1:2017  procedurg at 4 °C. The latter addition of low concentration of ISPC guarantees that putative DNA losses during extraction are additionally detected in individual samples [The method followed a previously established protocol ,27. In b samples . The supC. jejuni NCTC 11,168 or C. sputorum DSM 5363 (ISPC). Standards were used in duplicates in a set of serial dilutions  per reaction to generate standard curves for quantification. The National Reference Laboratory for Campylobacter provided the genomic standards in dried stabilized DNA aliquots as described previously [The targets of the qPCR assay have been previously described . Each qPeviously .TM Taq DNA Polymerase . The v-qPCR program started with a 15 min incubation at 95 °C, followed by 45 cycles of 30 s at 95 °C and 1 min at 60 °C (measure fluorescence) and 30 s at 72 °C for PlatinumTM Taq DNA Polymerase.The triplex v-qPCR method was employed using the fluorophore combination Jos-P-FAM, Csput-P-Cy5 and IPC-ntb2-P-HEX . The tri® qPCR ToughMix®  was employed as recommended. The triplex qPCR method is effective for most samples, but in cases where Campylobacter spp. exceed the maximum limit of quantification , it can hinder the ISPC signal and render it unsuitable for quantitative analysis [In cases where amplification was putatively hindered by elevated levels of humic acid, the PerfeCTaanalysis . In thosanalysis .U-test (two-tailed) and Kruskal–Wallis test with Dunn’s multiple comparison test were employed. Differences were statistically different when p < 0.05 (**** p < 0.0001).Values are calculated as the mean (M) with a standard deviation (SD).All quantitative data were compiled in a Microsoft Excel spreadsheet. Statistical data analysis and graphs were created using GraphPad Prism 9.1.0 (221) . Mann-Whitney Campylobacter spp. Of these, Campylobacter spp. was detected only at four time points , while at visits 3–5, all samples (n = 12) from the barns tested negative for Campylobacter independent of the applied detection methods. Furthermore, no cultivable C. jejuni (CFU) or total C. jejuni DNA  was found in the environment (n = 72) at visits 3–5. Overall, 15.9% (13/86) of the environmental samples, were confirmed to be positive for viable C. jejuni cells when the barns were positive for C. jejuni, which corresponded to log10 viable C. jejuni (Cj)/sample after PMA treatment. Cultivable C. jejuni were confirmed in only one out of 86 environmental samples (water) (1.2%). Viable C. jejuni was not detected in any of the air samples (n = 28). However, when air samples were tested for C. jejuni DNA without PMA, all 28 samples (100%) were positive with a concentration of 2.5 ± 1 log10 dead Cj/m3 (p <0.0001). In boot swabs from the environment (n = 24), viable C. jejuni was determined in 11 samples (45.8%) at a concentration of 3.2 ± 0.8 log10 viable Cj/boot swab sample (p <0.0001). In environmental gauze swabs, C. jejuni DNA from dead cells was identified in 10 samples (n = 18), with a concentration of 2.5 ± 0.2 log10 dead Cj/gauze swab sample (p < 0.0001).In total, seven sampling time points were analyzed for the presence of C. jejuni was identified in one gauze swab at a concentration of log 1.5 log10 viable Cj/gauze swab. However, this result, along with a positive finding in one water sample of 1.8 log10 viable Cj/water sample (n = 16), was below the validated limit of quantification (LOQ of 2 log10) of the method and, thus, interpreted as semi-quantitative values. Nevertheless, equal quantities were cultivated in the water sample (1.5 log10 CFU/water sample). Regarding barn matrices, , providing an accurate estimation by PMA despite potential negative matrix effects. Strikingly, Campylobacter was not cultivated from one chicken manure sample while simultaneously v-qPCR determined 3.5 log10 viable Cj/5 g manure. The corresponding boot swab sample, probably soiled with fresh fecal or cecal droppings, contained ~6.5 log10 viable C. jejuni with v-qPCR and CFU per boot swab  were collected after removal of the flocks from separate experimental animal rooms and analyzed using CFU and v-qPCR. The CFU method yielded 0 CFU/g (n = 16) of C. jejuni in all examined manure samples immediately after the removal of the flocks (sampling point 0). In contrast, the qualitative detection of C. jejuni using enrichment culture yielded positive results in the samples. Cultivable C. jejuni was detected for up to 24 h by qualitative detection methods. Interestingly, v-qPCR revealed that viable C. jejuni was quantifiable in most of the pre-treated samples at time point 0, with concentrations of 3.7 log10 ± 0.8 viable Cj/5 g manure (n = 16) (10 viable Cj/5 g manure (n = 9) were still determined.Based on the results of the field trial, an experimental investigation was conducted to further analyze (n = 16) . Through(n = 16) . After 7Campylobacter was determined in soil in open and closed containers in the laboratory at room temperature (RT) (A), in an incubator at high humidity at RT (B) or in the refrigerator at 4 °C at ambient humidity (C. jejuni strain BfR-CA-14430 rapidly lost its cultivability in open containers under laboratory conditions at RT (A), thus being additionally exposed to daylight  were observed using containers with a closed lid after 24 h. The inactivation rate in soil using a closed container in habitat A was consistent with the inactivation rate in the incubator setting (B), regardless of whether it was stored in an open or closed container (C. jejuni demonstrated a notably longer cultivation period and higher quantities under refrigerator conditions (C) (10 CFU/g (n = 3)), and qualitative detection for up to 11 days. Using a closed container, cultivation capacity was further extended in habitat C, with 6.9 ± 0.16 log10 CFU/g (n = 3) detected on day 11. Afterward, cultivation capacity rapidly declined in habitat C, as well and quantitative cultivability was achieved for the last time on day 14  . Using aions (C)  (2.2 ± 0y 14 see .Campylobacter generated in raw milk was investigated. Viable but not culturable cells of C. jejuni BfR-CA-14430 were introduced into two different matrices (water and soil) at an approximate initial concentration of ~5 log10 viable Cj/g as determined by v-qPCR. Under condition A, viable C. jejuni were recovered from soil at a concentration of 3.46 ± 0.37 log10 viable Cj/g (n = 3) after 24 h. Within five days, viable counts decreased gradually by an additional ~1.5 log level to 2.17 ± 0.18 log10 viable Cj/g (n = 3)  to 1.96 ± 0.12 log10 Cj/g (n = 3). Moreover, condition B extended the stability in soil to day 11 (1.82 ± 0.17 viable Cj/g). In contrast, under condition C, an initial recovery of viable cells was detected after 24 h of 3.86 ± 0.2 log10 viable Cj/g (n = 3), while an average of 2.7 ± 0,3 log10 viable Cj/g was detectable until the end of the trial (day 25) in soil. In water, the introduced 5 log10 viable cells were detectable for up to 25 days in conditions A and B. In habitat C, on the other hand, there was no relevant decrease in viable cells even during prolonged storage over 109 days, when no viable Cj could be retrieved under conditions A and B  I. Similand B see .C. jejuni BfR-CA-14430 to achieve an initial concentration of approximately ~8 log10 CFU/g soil or ml water. Then, the investigation aimed to explore the loss of cultivability and the possible transition into VBNC. As observed in the tenacity study, the initial recovered amount (d0) was 7.0 ± 0.8 log10 CFU/g (n = 3) in soil. Under conditions A, CFU decreased rapidly after one day (d1) to 3.58 ± 0.23 log10 CFU/g when compared to the other microhabitats . Thereafter, C. jejuni could only be qualitatively detected after 48 h (d2) in habitat A in soil . Equivalent amounts could be detected until the end of the experiment on day 21  viable counts were detected by v-qPCR. Viable counts were continuously detected until day 21. Furthermore, CFUs were determined in soil until day 12 in habitat C. With the loss of cultivability after day 15, viable (3.97 ± 0.2) log10 viable Cj/g (n = 9) were determined) counts in habitat C with v-qPCR, which were stable until the end of the experiment (d28)  were spiked with cultivable  in soil I. After n day 21 I. In connt (d28) I.C. jejuni in water (habitat A) was observed. By day two (d2), only one of the three replicates showed a detectable level of 2.2 log10 CFU/mL of cultivable C. jejuni. Subsequently, qualitative detection was last possible in one replicate on day 3. However, the v-qPCR analysis (C. jejuni cells (7.9 ± 0.05 log10 viable Cj/g (n = 3). The gradual decay of viable cells by ~4 log10 was observable using v-qPCR until day 63  were still determined. In habitat C, the conditions preserved viable cells until day 63. Remarkably, under these conditions, C. jejuni remained cultivable for approximately three times longer compared to the other habitats, as shown before. Specifically, culturable C. jejuni were observed until day 14, and viable cells were detected until day 63 in water under condition C at cooling temperature  could be retrieved as CFU. In contrast, the absence of Campylobacter DNA (from dead and viable cells) in the environment of broiler farms correlated with the absence of cultivable Campylobacter in the barns. Campylobacter was primarily absent during the winter. This seasonal phenomenon was previously described [Campylobacter was infrequent, these recent data show VBNC Campylobacter presence which suggest Campylobacter transmission into the environment. Specifically, the determination of viable C. jejuni cells with v-qPCR in one water sample with simultaneous observation of lower CFU loads indicated a potential transition into the VBNC state. Therefore, it is reasonable to hypothesize that water bodies favor prolonged persistence while supporting the gradual transition of cultivable cells to the VBNC state. In support, reduced environmental stressors, such as low levels of dissolved oxygen and UV-light exposure in water, could be promoting longevity and thus a gradual transition of Campylobacter in the VBNC state [Campylobacter persistence [C. jejuni were primarily detected by v-qPCR after treatment with PMA in sock swab samples (45.8%), collected by walking a predetermined route at Campylobacter-positive farms. This phenomenon could potentially be elucidated by the presence of broiler manure residues in the surroundings, along with boot swabs frequently being contaminated with chicken manure. It is conceivable that contaminated manure is spread by personnel and their vehicles after partial depopulation, as this practice has been recently connected with Campylobacter transmission at broiler farms [Campylobacter DNA from dead cells was determined in gauze swabs, although one gauze swab was semi-quantitatively positive for viable C. jejuni with v-qPCR. This could be due to the increasing deposition and accumulation of ventilated broiler sheds and manure in the environment [Campylobacter was not determined in air samples (only Campylobacter DNA from dead cells was determined). These findings show the discrepancy between viable and dead cells, providing a new insight as previous research only utilized PCR methods without PMA and the ISPC [C. jejuni  were observed more frequently during periods of ‘mild weather’ characterized by overcast, cloudy, and rainy conditions .Campylobacter in the environment. The investigation of natural contaminated chicken manure from experimental pens aimed at the detection of VBNC C. jejuni in a partially controlled environment where both, the inoculation strain and the conditions of the experimental animal rooms were known. Overall, using cultural quantitative analysis, cultivable C. jejuni were not determined, although cultural qualitative detection was possible through enrichment directly after the removal of the flocks (0-h mark). This observation is consistent with previous research suggesting that Campylobacter may rapidly lose its cultivability in manure [C. jejuni counts, comparable to those obtained in the field trials of barns (~3.5 log10 viable Cj/5 g manure), indicating that any potential bias introduced by physical pretreatment of the samples was at least reproduced. These results indicate that VBNC C. jejuni remained stable in chicken manure for several days. Indeed, these observations confirm the hypothesis from the field trial, and substantiate the assumption that VBNC Campylobacter originating from manure can be released directly from the animal barn into the environment. Nevertheless, it is important to reiterate that the storage of the manure was carried out under controlled and stable conditions, within an experimental animal room. These conditions encompassed factors like: the absence of UV light exposure, variations in air exchange rate, and relative humidity, which could differ from the conditions found in the natural environment.During the field trial, chicken manure and water were found to be two conceivable reservoirs of VBNC n manure . More imn manure ,42. ConsC. jejuni strain in soil (placed in open (with desiccation) and closed containers (without desiccation)) were determined under laboratory conditions (A). In open containers, a rapid loss of C. jejuni cultivability (within one day) was observed at 21 °C and low RH, which was anticipated due to desiccation stress caused by low humidity [C. jejuni was detectable by cultural detection methods for up to three days under laboratory (A) and incubator (B) conditions when using high relative humidity (99%). Desiccation appeared to be an important environmental driver for C. jejuni survival as its capacity was reduced by a loss of moisture, which might additionally lead to increased oxygen tension. Similarly, shifts in temperature were shown to correlate with C. jejuni viability. In particular, low temperatures around 4 °C were observed to extend the survival capacity of C. jejuni in soil. This observation is substantiated by an extensive body of research that has noted prolonged survival in both water and food matrices at 4 °C [First, the natural decay and loss of cultivability of the humidity . In cont at 4 °C ,44,45,46C. jejuni BfR-CA-14430 (induced in raw milk) was investigated in the same microhabitats. Despite observing cell losses during the recovery process from the soil matrix, as discussed above, VBNC C. jejuni were still detected for an extended period, depending on the utilized microhabitat. However, it should be noted that soil components themselves, such as humic acid, may interfere with qPCR and may have a negative impact on qPCR [C. jejuni target. Hence, it was manageable to address this issue by implementing a more resilient DNA polymerase for the soil samples. This adjustment allowed us to accurately assess inactivation through PMA monitored by ISPC. Remarkably, within the laboratory environment (A), desiccation and oxygen stress had a less severe impact on the integrity of C. jejuni cells that were already in the VBNC state. Despite the lack of desiccation and moisture loss (induced by inoculation) in habitat (B), maintaining a relative humidity of 99%, the initial higher levels of viable cells observed in comparison to habitat (A) experienced a subsequent 2-log reduction by the end of the 11-day trial period. This decrease could be linked to other biotic factors within the soil matrix at the temperature range of 21–22 °C. At refrigerator conditions (C), however, viable C. jejuni cell counts remained stable for prolonged periods, again indicating that low temperatures are favorable for VBNC C. jejuni. Interestingly, storing milk-induced VBNC C. jejuni in water resulted in maintenance of viable C. jejuni counts during the experimental period of 25 days in habitats A and B and even 109 days in habitat C. This may imply that stress conditions in water have less impact on VBNC stability than those encountered in soil [Campylobacter in both water- and food-related matrices under comparable conditions [Second, the stability of VBNC  on qPCR . These e in soil ,48. Thesnditions ,21,22,33C. jejuni into VBNC C. jejuni in soil and water was observed. Under laboratory conditions (A), rapid loss of cultivability in soil was observed as previously described. Thereafter, viable C. jejuni was detected using v-qPCR until day 28. Notably, the inoculation strain also lost its cultivability very rapidly in water (within three days) at RT. Overall, a ~4 log reduction of viable C. jejuni cells was observed in water samples over time at any condition but slower at refrigerating temperature. However, cells remained viable until the end of the investigation on day 63. It might be assumed that the exposure to daylight under condition A in open containers, filtered by glass windows, might have harmed viable cells over time due to the sensitivity of bacteria to photo-oxidative damage [C. jejuni were detectable in both water and soil for up to five days by qualitative detection, suggesting that the matrix itself was negligible at high humidity. Moreover, viable C. jejuni cells were detectable until the end of the trials (in soil until day 28 and in water until day d63). These results are in line with prior observations of in milk-induced VBNC C. jejuni, which corroborates the aforementioned studies.Finally, the gradual transition of culturable e damage . Under iCampylobacter can persist in contaminated environmental matrices under favorable conditions. In the subsequent experimental trials, prolonged stability of viable C. jejuni in water was demonstrated by v-qPCR, especially at 4 °C. Therefore, it is of major interest to understand the potential risk of water-associated VBNC C. jejuni in the agricultural environment. Several studies assessed C. jejuni in different waters using qPCR [C. jejuni. Thus, employing v-qPCR in conjunction with ISPC could offer additional insights into the presence of VBNC-Campylobacter in aquatic farm environments, as previously indicated [C. jejuni in primary chicken embryo cells (accordingly outgoing pathogenicity) was demonstrated [C. jejuni may persist in manure residues or contaminations in the environment. Consequently, it is feasible that VBNC-Campylobacter are transmitted via contaminated manure that remains in the environment after flock removal and are introduced into subsequent flocks [C. jejuni in naturally contaminated manure from experimental pens for up to 72 h. It can be assumed that the transition into VBNC states within manure may pose the challenges encountered in cultivating Campylobacter from chicken manure [Campylobacter detection in manure by cultivation, as observed in this and previous studies [C. jejuni were found. It is possible that various abiotic factors in soil, such as desiccation, physical entrapment, fluctuating oxygen levels, and biotic factors like competition from soil microflora, may be potential drivers of rapid VBNC formation, which resulted in, on the one hand, faster formation of VBNC Campylobacter and, on the other hand, reduced stability compared to water [The results of the field study show that VBNC ing qPCR ,50,51,52ndicated . The urgnstrated . As desct flocks . Furthern manure . Indeed, studies . This mi studies  found thto water .Campylobacter in the environment and under predefined conditions. Utilizing v-qPCR for the analysis of samples obtained under favorable environmental conditions , we were able to display viable C. jejuni and, thus, potential VBNC Campylobacter in the environment of broiler farms. The results of the experimental trials showed that viable C. jejuni cells could be detected in manure for up to 48 h. In our laboratory experiments, we were able to demonstrate that VBNC Campylobacter can remain viable over extended periods in different evaluated settings. Specifically, VBNC Campylobacter was viable for prolonged periods in water. However, it is important to note that controlled experimental settings do not necessarily reflect and mimic the VBNC Campylobacter persistence in nature (the environment). While we found that temperature, desiccation, humidity, and UV light appear to be important environmental drivers for VBNC Campylobacter, it should be emphasized, however, that other factors such as strain-specific differences and nutrient availability may also influence the persistence of VBNC Campylobacter. It is important to note that PMA alone confirms cell membrane integrity but provides no data on metabolic activity, pathogenicity, or infectivity. To gain deeper insights into VBNC states, further concurrent studies with v-qPCR, colored staining, microscopy, and in vivo/in vitro assays using isolated VBNC Campylobacter are of particular interest. To conclude, this comprehensive approach enhances and elucidates the overall understanding of the role of VBNC Campylobacter in the poultry reservoir.This study aimed to investigate the transition and persistence of VBNC