Background: Overuse and misuse of antibiotics contribute unnecessarily to antibiotic resistance (ABR), and are thereby global health threats. Inappropriate prescriptions of antibiotics during pregnancy, delivery and early childhood are widespread across the world. This study aimed to assess knowledge, attitudes, and reported practices of healthcare providers (HCPs) and to explore their perceptions regarding antibiotic use and ABR related to pregnancy, childbirth, and children under two in Lao PDR. Methods: This is a mixed methods study with data collection in 2019 via structured interviews among 217 HCPs , who prescribed/dispensed antibiotics in one rural and one urban district in Vientiane province and individual qualitative interviews with 30 HCPs and stakeholders. Results: Of the HCPs, 36% had below average knowledge regarding antibiotic use and ABR, and 67% reported prescribing antibiotics for uncomplicated vaginal delivery. Half of the HCPs did not believe that their prescribing contributed to ABR, and only 9% had participated in antibiotic education. Conclusion: A substantial number of HCPs had suboptimal knowledge and prescribed antibiotics unnecessarily, thereby contributing to ABR. Continuous education and regular supervision of HCPs is recommended to improve the use of antibiotics related to pregnancy, childbirth, and young children. Antibiotics are lifesaving interventions allowing management of infections during pregnancy, delivery and the postpartum period, leading to reduced maternal and infant mortality . HoweverMaternal infections in pregnancy are common and several pre-existing conditions such as malnutrition, diabetes, obesity, severe anaemia, bacterial vaginosis and group B streptococcus infections, as well as pre-labour rupture of membranes, and caesarean sections may enhance the risk . Thus, wAlthough the use of antibiotics for uncomplicated vaginal delivery is generally not recommended by the World Health Organization (WHO) , high usTherefore, healthcare providers (HCPs) taking care of pregnant women, childbirth and children play a crucial role in avoiding unnecessary use of antibiotic. Every time doctors prescribe antibiotics to patients, they need to have a good knowledge regarding antibiotic to assure rational use and to avoid possible complications. Previous studies demonstrated that HCPs’ attitudes and knowledge determine the quality of prescription of antibiotics, and that understanding of HCP’s knowledge, attitudes and practices towards antibiotic use and ABR is a key to develop behaviour change interventions ,25. It hHowever, data on antibiotic use during pregnancy and childbirth is scarce in Laos and little is known about HCPs’ knowledge, attitudes, perceptions and practices regarding antibiotic use and ABR, but it is highly relevant to the nation as a response to the challenges described above. The aim of this study was to assess HCPs’ knowledge, attitudes, and practices and to explore their perceptions regarding antibiotic use and ABR in relation to pregnancy, childbirth, and children less than two years of age in two provinces of Laos. Our study, by providing essential baseline data, has implications for planning of future interventions aiming to improve antibiotic use in Laos, and in the long term to contain ABR. It is also relevant to many other settings with similar situations.In total, 217 HCPs participated in the quantitative part, including 107 participants in Feuang district  and 110 in Vangvieng district (urban area) in Vientiane province. There were no statistically significant differences between the two study sites regarding participants’ characteristics, except for median age and distribution of HCPs across practice types. Compared to Feuang district, HCPs in Vangvieng were significantly older and worked in lower proportion at health centres (26% vs. 55%) and in higher proportion (65% vs. 39%) at district hospitals .Most of the HCPs in both study districts could without any problems identify antibiotics from a presented matrix of various medicines. All participants (100%) correctly classified ampicillin, amoxicillin, and penicillin as antibiotics; however, only 68% recognised that cortisone is not an antibiotic. The majority of participants (97%) agreed that some antibiotics may have teratogenic effects and should be carefully considered before prescribing to pregnant women. The proportion of participants who replied that antibiotic use may cause side-effects and can contribute to ABR was significantly higher in Vangvieng than in Feuang district (100% vs. 94% and 94% vs. 85%).The majority of participants (94%) had heard the term “antibiotic resistance”, knew that extensive use of antibiotics in society increases the risk of development and spread of resistance (91%), that the unnecessary use of antibiotics can make them ineffective in long term (89%), and that antibiotic course cannot be interrupted in the middle, even when symptoms of sick patients are improving (76%). Around one-third of participants (32%) replied that people cannot become resistant to antibiotics. The majority of participants (80%) agreed that today ABR is a big problem in their practice, 67% agreed that ABR is a problem in Laos and 58% that it is so worldwide .The majority of participants (93%) agreed with the statement “patient non-adherence to the therapy is contributing to ABR”. Half of the HCPs thought that their prescribing practice is not contributing to ABR. There were no statistically significant differences between the two study districts regarding participants’ attitudes towards antibiotic use and ABR .Eighty-five percent (184/217) of participants replied that they prescribed/dispensed antibiotics to a patient during the previous week, and of those, 67% (n = 123) had prescribed/dispensed antibiotics more than four times. Antibiotics were prescribed/dispensed in the highest proportions for fever (69%), sore throat (63%) and cough (51%). Tonsillitis (44%), pneumonia (15%) and pharyngitis (10%) were the most common indications for which antibiotics were given. The most common antibiotic prescribed was amoxicillin, penicillin (phenoxymethyl penicillin), ampicillin, and erythromycin. The majority of patients (65%) received antibiotics for 5 days, 25% for 7 days and 8% for 3 days. Among other medicines, paracetamol was the most commonly prescribed/dispensed (92%).Around two-thirds (67%) of all participants replied that they would prescribe/dispense antibiotics for uncomplicated vaginal delivery without episiotomy. The proportion of HCPs prescribing/dispensing antibiotics for this indication was significantly higher in Feuang than in Vangvieng district (81% vs. 54%).Sixty-five percent of participants reported that they followed the common practice in their health facility to prescribe/dispense antibiotics, and 23% explained that (even when it was not necessary) they prescribed because of the pressure from the patient. The proportion of HCPs prescribing/dispensing antibiotics due to concern about patients getting an infection was significantly higher in Feuang than in Vangvieng district (92% vs. 83%). Conversely, the proportion of antibiotics prescribed/dispensed due to pressure from patients was significantly higher in Vangvieng than in Feuang district (29% vs. 16%). Only a small fraction of all HCPs participated in antibiotic education activities and the proportion was significantly higher in Feuang than in Vangvieng district (13% vs. 5%) .The total scores of participants’ knowledge and reported practice regarding antibiotic use and ABR were above average  in 64% and 41% of all participants, respectively. There were no statistically significant differences in the knowledge and practice scores regarding antibiotic use and ABR between the two study districts .In total, 30 HCPs and key stakeholders at different levels of health facilities in Vientiane province and Vientiane capital participated in the individual qualitative interviews, including nine participants at central level, nine at provincial level, and 12 at district level. The participants comprised nine specialists (six obstetricians-gynaecologists (OG) and three other specialists), six medical doctors (MD)/assistant doctors (AD), nine midwives (MW)/nurses (N), and six pharmacists (P)/assistant pharmacists (AP). The participants held various positions including directors of provincial hospital/provincial health department/district hospital/district health office, heads of health centres, obstetrics-gynaecology/antenatal care (ANC) team leaders and other technical staffs. The mean age of participants was 44; 67% were females; 93% were Lao ethnic, and 7% were Hmong. The findings from the individual interviews are presented under each main theme with quotes from the participants to illustrate the findings. Three main themes were identified as follows: (i) perceptions of antibiotics prescribing; (ii) experience with ABR and patient-doctor interaction and communication; and (iii) opinions about sources of information and educational intervention on antibiotic use in relation to pregnancy, childbirth and children under two.The majority of participants stated a wider range of indications such as respiratory infection, urinary tract infection, or reproductive tract infection, as well as prophylactic provision in cases of caesarean section or uncomplicated vaginal delivery with episiotomy to prevent post-partum infection.“We prescribe antibiotics for all cases before performing caesarean section, delivery with episiotomy cases, perineal tear or premature rupture of membranes to prevent post-partum infection”Some participants mentioned that the main reason for giving antibiotics to women with uncomplicated vaginal delivery was lack of confidence in proper sterilisation of delivery room and equipment. Moreover, HCPs doubted that patients understood well the provided instructions on how to manage wounds after the discharge.“The main reason that I prescribe antibiotics to pregnant women is to prevent and treat infection in uncomplicated vaginal delivery cases because we are afraid that the delivery room in the hospital is not guaranteed to be sterile”“In cases of uncomplicated vaginal delivery, during discharge from the hospital I prescribe amoxicillin tablets for 5 days to prevent post-partum infection as I am afraid that the patients did not understand properly about our health education provision regarding personal hygiene to keep their wounds clean”Only few participants stated that they did not prescribe any antibiotic during uncomplicated vaginal delivery.“In cases of uncomplicated vaginal delivery, I do not prescribe any antibiotics, following the guidelines of the WHO that the use of antibiotics is not recommended for uncomplicated vaginal delivery”The majority of participants mentioned that they always prescribed/dispensed antibiotics for children with respiratory infections such as pharyngitis, tonsillitis, or pneumonia. Nearly half stated that they prescribed/dispensed antibiotics to children with skin infection, wounds, or diarrhoea.“For children under two, I dispense antibiotics in case of respiratory infection such as pharyngitis, tonsillitis, skin infection, leucocytosis, trauma or others”“The majority of children received antibiotics because of diarrhoea, which is a seasonal disease that mostly occurred in summer”Half of participants reported that they observed ABR in their daily practice and thought that it was a health problem; they were concerned about patients commonly self-medicating with antibiotics to treat e.g., fever or cough before seeing a doctor. “I think that antibiotic resistance is a health problem. Sometimes patients self-medicate with antibiotics when they get fever; if they could not recover then they come to see doctors. Since bacteria are resistant to antibiotics, doctors need to prescribe higher doses of antibiotics”Few participants mentioned that they were not sure if bacteria are resistant to antibiotics, as they did not perform bacterial culture to confirm AMR in their health facility.“It is not easy to identify if bacteria are resistant or not, because we did not do investigations of drug resistance. However, there was a report about bacteria that caused Typhoid fever were resistant to antibiotics. For other bacteria I had never heard”Almost all participants declared that they provide information on antibiotic properties, use, possible adverse events and other relevant issues. However, a few participants said that they do not inform patients about bacterial resistance, as they themselves need to gain more knowledge.“If the result of blood test (such as leucocyte count) show bacterial infection we will explain to patients about which kind of antibiotics to be given, what are their properties, how much doses, why we need to use this drug. So we need antibiotic knowledge to explain to patients about the effectiveness of antibiotics, their mechanism of action, how to make patients feel safe on the treatment”Half of participants perceived that patients had some knowledge about antibiotics from social media or drug advertising. Others thought that patients did not have any knowledge about antibiotics and proposed that a majority of patients believed that antibiotics could treat a range of infections including a common flu or cough. They also cautioned that some patients may not complete the course of antibiotic. Few participants thought that antibiotic knowledge of patients depend on the level of education.“The majority of patients including pregnant women asked to buy antibiotics when they got flu or cough, as they thought that antibiotics can treat every illness. I thought that patients didn’t know how to use antibiotics for instance when they got sick and went to see doctors, who prescribed antibiotics to them for 5–7 days, but they took only 2–3 days then stopped using the medicines. We need to explain to patients that inappropriate use of antibiotic may cause antibiotic resistance”“I think that antibiotic use depends on patient’s knowledge. Some patients knew well how to use antibiotic and followed the doctor’s recommendation, but some had difficulty to understand as we have to explain to them many times how to take antibiotic, especially for minority ethnic group”Several participants stated that their source of information about antibiotics were national treatment guidelines, the WHO guidelines, lectures or training, magazines, scientific journals, or internet. “The majority of information on antibiotics that I got was based on national treatment guidelines, and the guidelines from WHO”“I received health information on antibiotic use from doctors who gave lectures based on the guidelines of paediatrics and obstetrics-gynaecology associations”The majority of participants stated that guidelines on antibiotic use during pregnancy, delivery and for children under two would be valuable for their daily practice and would allow them to provide correct treatment and sufficient health information to patients.“It would be good if antibiotic guideline is available, as we can follow the guideline how to treat, no need to ask for help from others, no need to try to get other sources of information on antibiotic use”“If antibiotic guideline is available it would be able to give correct information to pregnant women to understand better”Most of the participants mentioned that they did not receive any specific training about antibiotic treatment in relation to pregnancy, childbirth and children under two. Only a few participants had attended training on rational medicine use and other topics.“It would be great if continuing professional development is available. In the past, there was no training program regarding antibiotic use. The training on mother and child health education did not focus on antibiotic use, but rather focus on the health care during pregnancy, delivery and postnatal care for mother and child”Most of participants stated that they would like to attend training on antibiotic use in relation to pregnancy, childbirth and children under two, as they believed that it is the best way to improve their knowledge.“To improve HCPs’ knowledge, we need to attend training with trainers from the Ministry of Health to upgrade our knowledge on antibiotic use and other topics as well”About half of participants said that they would like to improve patients’ knowledge by providing health information through training, advertisements, posters and brochures.“It would be better to educate general population or pregnant women regarding antibiotic use in relation to pregnancy, delivery and children under two by using advertising, posters, brochures, then training HCPs”One participant said that training on antibiotic guidelines should be included in the medical university course. Another participant mentioned that advertising via social network is an option for educational intervention.“I think that we have to add antibiotic training into the learning program in the medical university to have more knowledge about antibiotic use before graduating”“I request to have posters, magazine, broadcast via radio about antibiotic use in pregnancy, childbirth and children under two or via social media like Facebook”To the best of our knowledge, this is the first study conducted in Laos that aims to assess knowledge, attitudes, practices of HCPs and to explore their perceptions regarding antibiotic use and ABR in relation to pregnancy, childbirth, and children under two. We found that one-third of HCPs had low knowledge about antibiotic use and ABR, two-thirds reported non-indicated antibiotics prescribing for uncomplicated vaginal delivery, and half of HCPs did not believe that their prescribing practice contributed to ABR. Importantly, the majority of HCPs had never participated in any antibiotic education activities. We observed significant differences in non-indicated prescription between the study areas with a higher proportion of antibiotic prescribed for uncomplicated vaginal delivery in Feuang than in Vangvieng district (81% vs. 54%). The reason was that the majority of HCPs, especially in the rural area, was concerned about patients developing infection-related complications. This was evident from our qualitative findings, where HCPs explained that the main reason for prescribing antibiotics in case of uncomplicated vaginal delivery was to prevent post-partum infection linked to the fear that the sterilisation of delivery room and equipment was not guaranteed and that the health education provided would not be well understood by patients. The results are in line with a previous study conducted in Laos in 2008 presenting that 79% of women who had uncomplicated vaginal delivery received antibiotics in hospitals . StudiesThe findings of our study revealed that 36% of HCPs had below average knowledge regarding antibiotic use and ABR and the level was comparable between the rural and urban area. This might be due to participants’ similar demographic characteristics including types of HCPs in both study districts. In addition, they underwent the same education and, as presented in the qualitative findings, received information on antibiotics from the national treatment guidelines, the WHO guidelines, lectures or training, magazines or scientific journals and from the internet. Although the majority of all participants reported that antibiotics are effective against bacterial infections, about one-third thought that common cold is normally caused by bacteria; this may contribute to HCPs’ decision to prescribe antibiotics to patients with a common cold. Previous studies from other countries also showed that about 20–60% of the participants believed that antibiotics should be prescribed for cold of viral aetiology ,30,31. KAn interesting aspect was that half of our participants did not think that their own prescribing affected ABR; this could result in prescribing behaviours that actually promoted ABR. It has been shown that the majority of participants declared that the previous use of antibiotics increased the person’s risk of acquiring drug-resistant infection . In addiOur findings showed that nearly two-thirds of all participants reported that they prescribed antibiotics by following the common practice in their health facility. One-quarter prescribed antibiotics due to the perceived pressure from the patient, although the HCPs did not think it was necessary. Additionally, our qualitative findings showed that half of the HCP participants mentioned that the majority of their patients believed that antibiotics can treat every illness and want to use antibiotics even in cases of flu or cough. Studies from other countries also reported that most physicians identified that they felt being under pressure if the patients expected an antibiotic prescription, even for treating viral infections; this may contribute to antibiotic overuse and ABR ,32,34. IOnly 9% of HCPs reported that they had participated in antibiotic education. This was confirmed during the qualitative interviews, when most of the participants mentioned that they did not receive any specific training about antibiotic treatment and that the continuous professional development was not in place. Most of HCPs requested to have more educational programs regarding antibiotic prescribing, especially in relation to pregnancy, childbirth, and young children, as they believed that it is the best way to improve their knowledge. Studies from other countries also showed that the majority of participants needed treatment guidelines and educational programs on antibiotics prescribing ,42. A stThe strengths of this study were that the quantitative data were triangulated with qualitative findings. The study has provided useful information on HCPs’ perceptions, knowledge, attitudes, and reported practices regarding antibiotic use and ABR. The quantitative and qualitative findings gained from this study can be used to develop educational interventions to promote appropriate antibiotic use. However, every study needs to be seen in the context of its limitations. First, the quantitative part was conducted in only two districts of Vientiane province, and although they represented different ethnic minority population and different settings , may not be representative of all the country. Second, all provided responses were self-reported and might not reflect the actual practice, since it was not possible to ascertain whether what the HCPs reported was an accurate description of their real practice of antibiotic use, thus, the possibility of reporting bias cannot be excluded. Third, all responses were based on memory of past events that may affect accurate recall, resulting in both under-and over-reporting.This is a mixed method, cross-sectional study containing quantitative and qualitative components. The presented study is a part of the CAREChild  project conducted in Laos . The study was carried out in Laos, a lower middle-income country in Southeast Asia with approximately 7 million inhabitants and relatively low population density . Laos haThe quantitative part of the study took place in Vientiane province in the middle part of Laos. Vientiane province has 11 districts with the total population of 450.000 inhabitants. Two districts (Feuang and Vangvieng) were purposively selected as study areas based on variation in participants background characteristics, representing different ethnic minority populations, and also for feasibility reasons. Feuang district is a rural area, and its health care system is composed of 1 district hospital, 6 health centres, 8 private pharmacies and 107 HCPs. Vangvieng district is an urban area and has 1 district hospital, 5 health centres, 10 private pharmacies and 110 HCPs . The qualitative part using individual interviews was carried out in Thoulakhom district in Vientiane province and Maternal Child-Newborn Hospital (MCNH) in Vientiane capital, which were outside of the study areas. Thoulakhom district was purposively selected for feasibility reasons; it has 1 district hospital and 5 health centres. The MCNH is 1 of the 5 central hospitals, and is a university hospital, where women from different parts of the country come for ANC and consultation. All HCPs  who prescribe or dispense antibiotics at the health facilities  and private pharmacies in both study sites were invited to participate in the quantitative study. They were identified from the health staff registry available at the district health offices. Representatives of HCPs and policy makers in Vientiane province , and representatives from central level such as Ministry of Health, University of Health Sciences, obstetrics-gynaecology association in addition to the MCNH in Vientiane capital were invited to take part in the individual interviews. They were purposively selected upon the identification in the local health staff name list.Structured questionnaires were developed based on previously used tools within and outside the project group and adapted for the different target groups to collect data on knowledge, attitudes and reported practices regarding antibiotic use and ABR in relation to pregnancy, delivery and early childhood . The stuA qualitative interview guide was developed and pre-tested in Phonehong district with 4 HCPs working across 1 district hospital, 1 health centre, and 1 private pharmacy to ensure that the questions were understood. This tool was used to explore HCPs’ perceptions towards antibiotics prescribing, ABR, patient-doctor interaction and communication, sources of information and educational intervention on antibiotic use in relation to pregnancy, childbirth and children under two. Based on the feedback from the pre-test the interview guide was adjusted and finalised. All data collection tools were developed in English, translated to Lao and pre-tested. Necessary adjustments were performed in Lao language. The final versions of the tools were then back-translated to English to check for consistency in the translation.Quantitative data was collected from September to November 2019. All included HCPs were informed about the purpose of the study and consented to participate. The interview was performed at a convenient place such as a meeting room or their office. Digital capture was used to collect data on social demographic characteristics, knowledge, attitudes, and reported practices of HCPs regarding antibiotic use and ABR. The individual qualitative interviews were carried out in the same time period by 1 interviewer and 1 note taker/audio-recorder, who were research team members. The participants were interviewed at the time and location of their choice, mostly at their office. Each interview lasted approximately 40 min. Sessions were continued until data saturation was reached (limited or no new information was obtained) .2-test) was used to compare two categorical variables, and Mann–Whitney was used to compare continuous variables, particularly comparing differences between Feuang  and Vangvieng district (urban area); a p value < 0.05 was considered statistically significant. Data were managed by using Research Electronic Data Capture (REDCap) tool hosted at Karolinska Institutet . Data weThe reported practice and knowledge scores were calculated based on correct (1 point) or incorrect (0 point) answer received from HCPs regarding their practice and knowledge. The points were summarised, and the mean score was calculated. If the total score was greater than the mean the reported practice and knowledge were classified as “above average” and if less or equal to mean they were classified as “below average”. The attitudes of HCPs towards antibiotic use and ABR were assessed by using a 5-point Likert like scale based on the statements whose responses included “strongly agree”, “agree”, “don’t know”, “disagree” and “strongly disagree”. We combined “strongly agree” and “agree” answers to define “agree”. We also performed the same combination for “strongly disagree” and “disagree” to define “disagree”.The audio-records and notes from the individual qualitative interviews were transcribed verbatim. The Lao transcripts were entered into the computer (as a word file) and analysed using qualitative content analysis . The valThis study indicated that one-third of HCPs had low ABR-related knowledge, two-thirds reported prescribing antibiotics for uncomplicated vaginal delivery, and half claimed their prescribing of antibiotics did not contribute to ABR. Unnecessary, non-indicated antibiotics prescribing coupled with low knowledge among a substantial number of HCPs may be an underlying reason for the development and spread of ABR. Therefore, continuous antibiotic education for HCPs and regular supervision are recommended in order to improve the use of antibiotics in relation to pregnancy, childbirth, and young children in health facilities.


Antibiotic consumption is increasing worldwide, particularly in low and middle-income countries (LMICs). Access to lifesaving antibiotics in LMICs is crucial while minimising inappropriate use. Studies assessing the economic impact of inappropriate antibiotic use in LMICs are lacking. We explored the economic impact of inappropriate antibiotic use using the example of upper respiratory tract infections (URIs) in Ghana, as part of the ABACUS (AntiBiotic ACcess and USe) project.A top-down, retrospective economic impact analysis of inappropriate antibiotic use for URIs was conducted. Two inappropriate antibiotic use situations were considered: (1) URIs treated with antibiotics, against recommendations from clinical guidelines; and (2) URIs that should have been treated with antibiotics according to clinical guidelines, but were not. The analysis included data collected in Ghana during the ABACUS project (household surveys and exit-interviews among consumers buying antibiotics), scientific literature and stakeholder consultations. Included cost types related to health care seeking behaviour for URIs. Additionally, cost saving projections were computed based on potential effects of future interventions that improve antibiotic use.Health care costs related to inappropriate antibiotic use for URIs were estimated to be around 20 million (M) USD annually, including 18 M USD for situation 1 and 2 M USD for situation 2. Travel costs and lost income due to travel, together, were estimated to be around 44 M USD for situation 1 and 18 M USD for situation 2. Possible health care cost savings range from 2 to 12 M USD for situation 1 and from 0.2 to 1 M USD for situation 2.This study indicates that inappropriate antibiotic use leads to substantial economic costs in a LMIC setting that could have been prevented. We recommend investment in novel strategies to counter these unnecessary expenditures. As the projections indicate, this may result in considerable cost reductions. By tackling inappropriate use, progress can be made in combatting antibiotic resistance.The online version contains supplementary material available at 10.1186/s13756-022-01096-w. Antibiotic resistance is an increasing global health emergency and leads to treatment failure of patients with infectious diseases . An impoTherefore, the AntiBiotic ACcess and USe (ABACUS) I project was conducted between 2016 and 2019 to explore community antibiotic access and consumption practices in Asia and Africa in order to identify targets that inform the design of interventions to improve antibiotic use , 7. FindGlobally, there were an estimated 4.95 million (M) deaths associated with bacterial antimicrobial resistance (AMR) in 2019, including 1.27 M deaths attributable to bacterial AMR . The futThe objective of this study is to explore the economic impact of inappropriate antibiotic use in the community setting in Ghana, related to upper respiratory tract infections (URIs) treated with antibiotics. It hereby fills an existing gap, as it is the first to provide such estimates for Ghana or any LMIC setting, and helps developing the economic case for sustainable investment in combating antibiotic resistance . The stuThis was a top-down, retrospective economic impact analysis focussing on inappropriate use of antibiotics for the treatment of URIs in the Republic of Ghana. We focused on URIs as this group of infectious diseases is an illustrative example for which inappropriate use in the community is common , 14. In Situation 1: URIs that were treated with antibiotics, against recommendations from clinical guidelines as considered self-limiting.Situation 2: URIs that should have been treated with antibiotics according to clinical guidelines as considered bacterial and sufficiently severe, but were treated with a painkiller.In Ghana, people with URI symptoms generally seek care at CDROs. All CDRO visits for URIs that lead to treatment that is not in accordance with antibiotic use guidelines, were reasoned to result in one of the following two inappropriate antibiotic use situations:We performed a separate economic impact analysis for each inappropriate use situation. A detailed overview of the included variables, values, and the information sources used for the analyses is shown in Tables For the analysis, we primarily collected unpublished data from the former ABACUS I project, originating from customer exit-interviews (~ 2300 interviewees) and household surveys (~ 1100 households visited) conducted in the Kintampo North and South districts, and from a newly conducted online survey using SurveyMonkey Inc. , or the use of diagnostics –36.Understanding the economic consequences of community antibiotic consumption practices is crucial to mobilise key stakeholders and design sustainable strategies to improve antibiotic use. This economic impact analysis supports this endeavour by providing baseline data to be used by future studies investigating the cost-effectiveness of such strategies. In a broader sense, the study can serve as a starting point for other countries, especially LMICs, to conduct an economic analysis related to inappropriate antibiotic use.The study has several limitations. First, it must be noted that inappropriate antibiotic use is a highly complex and comprehensive issue and providing a potential intervention is out of this study’s scope. Multiple factors can lead to URIs being treated in discordance with clinical guidelines. According to Godman et al. , sub-optThis study demonstrates that inappropriate antibiotic use leads to substantial economic costs in Ghana. These costs should be seen as unnecessary and preventable. We recommend investment in novel strategies to counter these expenditures. These interventions should be implemented in consultation with funders, industry, health care workers, pharmacists, policy makers, regulators, social scientists, and marketeers to ensure a successful roll-out. As the projections indicate, this may result in cost reductions. In addition, by tackling the inappropriate use problem, important progress can be made in combatting antibiotic resistance.Additional file 1. CHEERS checklist.Additional file 2. Expert survey - questions and responses.


Antibiotic free farms are increasing in the poultry sector in order to address new EU regulations and consumer concerns. In this pilot study, we investigated whether the efforts of raising chickens without the use antibiotics make any difference in the microbiome of poultry meat eaten by consumers. To this aim we compared the microbiomes characterizing caeca and the corresponding carcasses of two groups of chickens reared, one reared on a conventional farm and one on an antibiotic-free intensive farm. The results showed a clear separation between the taxonomic, functional and antibiotic resistant genes in the caeca of the birds reared on the conventional and antibiotic free farm. However, that separation was completely lost on carcasses belonging to the two groups. The antibiotic-free production resulted in statistically significant lower antimicrobial resistance load in the caeca in comparison to the conventional production. Moreover, the antimicrobial resistance load on carcasses was much higher than in the caeca, without any significant difference between carcasses coming from the two types of farms. All in all, the results of this research highlighted the need to reduce sources of microbial contamination and antimicrobial resistance not only at the farm level but also at the post-harvest one. In 2019 the European Union (EU) produced an estimated 13.3 million tonnes of poultry meat, representing an increase of around 27% in comparison to 2010 [There are different strategies to achieve antibiotic free poultry flocks such as the implementation of effective biosafety measures and management options as well as the promotion of beneficial microbes in the gastrointestinal (GI) tract of chickens to enhance animal health and inhibit pathogen colonization. To this aim, feed can be supplemented with probiotics and prebiotics, and also blended in the same supplement , to ensure diversity and stability of the GI microbial community, as well as positive interactions with the host’s gastroenteric epithelium and immune system . As an aIn the last decades, the microorganisms colonizing the chicken gut have been mainly investigated using classical microbiological cultural methods. However, the application of high-throughput sequencing techniques is now allowing a deeper investigation of the whole microbial composition of the chicken GI tract, including strictly anaerobic and not cultivable microorganisms. With reference to the high-throughput sequencing techniques, Durazzi et al.  comparedThe increasing incidence of antibiotic resistance is considered one of the major threats to global public health ,11. In tTo limit the use of antibiotics in animal rearing, Regulation (EC) No. 1831/2003  on VeterIn the pilot study described in this paper we investigated whether the efforts of raising chickens without the use of antibiotics made any difference in the microbiome of poultry that consumers eat. To this end, we compared the microbiomes characterizing caeca and the corresponding carcasses of two groups of chickens reared on different farms—one conventional and one antibiotic-free. Moreover, with the view of planning future studies, we investigated whether checking the correlation between the microbiome and resistome in the caeca and the carcass of the same animal provides more insight than the same analysis performed at flock level.A total of 30 slaughtered broilers were randomly sampled at the slaughterhouse within the first group of animals processed at the start of the working day, when the slaughterhouse was still clean and disinfected to avoid bias due to cross-contamination. Among the selected broilers, 15 belonged to a poultry flock reared in a conventional farm and treated with Amoxicillin (20–30 mg/kg of live weight in drinking water for three to five days) at days eight and 29 and with sulfadimethoxine/trimethoprim (100 mg/20 mg in 1–2 L of drinking water once a day for five days) at day 24. The remaining 15 belonged to a poultry flock reared in a conventional farm but never treated with antibiotics. All tested animals were female, ROSS, fed with no medicated feed and slaughtered at 47 (antibiotic free) and 48  days. For each sampled animal, both the gastrointestinal tract and the carcass were collected. The GI tract was sampled during slaughtering, at the evisceration step, and immediately stored in a sterile plastic bag kept at 4 °C in a refrigerated box. Moreover, a sterile plastic flag was attached to the hook transporting the carcass from which the GI tract was sampled in order to pick up that specific carcass at the end of the refrigeration tunnel. After sampling, each carcass was stored in a sterile plastic bag kept at 4 °C in a second refrigerated box, different from that containing the GI tracts. All samples were transported to the laboratory within two hours and immediately processed.g. The obtained pellet containing the concentrated cells was stored at −80 °C until DNA extraction.Each GI tract was dissected and a small portion  of caeca content was collected, transferred into a 2 mL sterile plastic tube, and flash frozen in liquid nitrogen before storage at −80 °C until DNA extraction. A total of 10 g of neck and breast skin were collected from each carcass, diluted in 90 mL of sterile physiological solution (NaCl 0.90%), homogenized in the stomacher  at normal speed for 1 min and centrifuged at 4 °C for 20 min at 9980× ® DNA Stool Mini Kit  for samples of caeca content [® Microbial DNA Isolation Kit (MO BIO-Qiagen) for the pellet obtained from each carcass [®  to assess DNA yield, in terms of quantity and quality. Total DNA from caeca and carcass samples was fragmented and tagged with sequencing indexes and adapters using the Nextera XT DNA Library Preparation Kit . Shotgun metagenomic sequencing was performed using the NextSeq500 (Illumina) 2 × 150 bp in paired-end mode. One caeca sample was removed later in the process for technical reasons (linked to sequencing yield) resulting in a total of 59 metagenomes: 29 from broilers caeca and 30 from carcasses.The DNA was extracted using a bead-beating procedure followed by the QIAmp content , and by  carcass . The DNAhttps://www.mg-rast.org (accessed on 14 December 2021) [Filtering and trimming of raw reads were performed using MG-RAST er 2021)  bioinforThe statistical analysis of both the taxonomic and functional gene composition was performed using R 3.6.3 and the libraries phyloseq 1.30.0  and DESeBefore proceeding with the statistical analysis, read counts were normalized with DESeq2 to take into account the different sizes of the samples. In this step, size factors were estimated using the function estimateSizeFactors of DESeq2 with the “poscount” option, as suggested when dealing with sparse data. Then, DESeq2 was used to assess whether the taxa/function abundances differed between groups. Specifically, the Wald test was used to determine the statistical significance and the Log Fold Changes were shrunk using the apeglm method . Finallyt-test to assess whether the linear relationship between alpha diversity and the grouping was negligible. Beta diversity was estimated starting from the read counts normalized with DESeq2 and computing the Bray-Curtis dissimilarity [Alpha diversity was estimated using the InvSimpson/Shannon/Chao1 index, and differences in alpha diversity between groups were evaluated fitting a linear regression model and using the Student’s milarity  among samilarity  to DESeqThe resistome of each sample was predicted using the Resistance Gene Identifier (RGI) . Fastq rhttps://www.mg-rast.org/mgmain.html?mgpage=project&project=mgp89213 (accessed on 14 December 2021) and described in Metagenomic sequencing yielded an average of 5 Gbp/sample. The 59 metagenomes sequenced are publicly available from MG RAST at p-value < 0.00001) (The beta diversity index (generated using the Bray-Curtis distance metric) calculated at the genus level highlighted a clear dissimilarity in the community composition in the caeca sampled in the two groups of broilers tested (adonis2 0.00001) .Alkaliphilus, Desulfibacterium, Bacillus and Ethanoligenens were only detected in the caeca of birds from the conventional farm   .Fourteen of the top 20 most abundant functional genes identified within the caeca of birds were shared between the two tested groups . MoreoveBesides these qualitative differences, the normalized mean values of beta-galactosidase (EC 3.2.1.23) and DNA topoisomerase III EC 5.99.1.2 (in PFGI-1-like cluster) were significantly higher in the caeca of birds from the conventional farm, while those of integrase, translation elongation factor G, ribonucleotide reductase of class III (anaerobic)-large subunit EC 1.17.4.2, excinuclease ABC subunit A paralog were greater in the Bacteroides group, and the chaperone protein DnaK was significantly higher in the caeca of birds reared in the antibiotic free farm .p-value = 0.020) according to the farm of origin   .Three of the top 20 most abundant functional genes identified on the carcasses were shared between the two tested groups . Moreovep < 0.05).As for the genera, the functional genes did not show significative different normalized mean counts between the two groups of carcasses tested  .The antibiotic resistance genes (ARGs) were retrieved from functional genes identified in the caeca and in the carcasses of the tested animals and classified as such in level 2 of the biological-function ontology in the SEED category . Among t< 0.000) . HoweverThe drug classes identified in the broiler caeca and on carcasses are listed in p-value = 0.00001)   , while i 0.4278) .Currently, conventionally raised poultry continues to dominate the EU poultry industry. However, there is an increasing consumer demand for meat obtained in antibiotic-free rearing cycles. Moreover, in January 2022, the new EU regulations of June 2019 [The results demonstrated three key findings. The first one is a clear separation between the taxonomic, functional and antibiotic resistance genes in the caeca of the birds reared on the conventional and antibiotic-free farms. This result is due to the fact that each poultry farm has an associated ecosystem due to the geographical and specific environmental conditions, to what chickens eat and drink, to the litter type, to the workers, and certainly to the medications they receive or not. That separation was completely lost on carcasses belonging to the two groups, which did not mirror whatever positive or negative impact the farm ecosystem and rearing conditions had on the chicken caeca. As for the caeca, and also for the carcass microbiomes, there are many contributing factors besides the possible cross contamination during the evisceration. Indeed, the ecosystems interacting with the animals during transport and then during each slaughtering step, including the final refrigeration tunnel, all contribute to the final carcass microbiomes.Escherichia coli than the historical use of antimicrobials at the farm level. Overall, these observations demonstrate that the effort of reducing antimicrobial use by means of rearing antibiotic free flocks should be associated with a better understanding of the antimicrobial resistance persistence in the farm environment in the absence of direct antimicrobial use. Further insights into the antimicrobial resistance persistence in the farm environment might help us to understand why, for instance, the relative abundance of acriflavine resistance protein genes was higher in the caeca of antibiotic-free animals compared to conventional ones.The second key finding is that the antibiotic free production resulted in statistically significant lower antimicrobial resistance loads in the caeca of chickens in comparison to the conventional production, thus confirming that besides external sources of ARGs, when antimicrobials are not administered to the animals in the caeca of that flock there is a lower antimicrobial resistance load. In relation to the short- and long-term effects of the use of antimicrobials on antimicrobial resistance, Mughini-Gras et al. 2021  showed tThe last main finding is that the antimicrobial resistance load on carcasses was much higher than in the caeca, without any significative difference between carcasses coming from the two types of farming. As described above, this result demonstrated that all post-harvest steps, including transport and slaughtering, but also the loading and unloading of the animals contributes not only to the microbiome colonizing the final carcass reaching the consumers, but also to its antimicrobial resistance load. Therefore, although the most important antimicrobial resistance risk factors and possible mitigation measures are still under investigation at the farm level , the impAlkaliphilus, Desulfibacterium, Bacillus and Ethanoligenens were identified as signature genera in the birds from the conventional farm, while butyrate-producing microorganisms such as Coprococcus, Roseburia and Subdoligranulum were identified in the caeca of birds reared on the antibiotic-free farm. This result highlighted that besides the higher bacteria biodiversity identified in the caeca of birds from the conventional farm, the signature genera colonizing the caeca of the birds reared on the antibiotic-free farm belonged to microbial groups supporting animal health. Indeed, butyrate fights against pathogen colonization in poultry [The alpha diversity calculated in this study at genus level using the indicators of richness (Chao1), evenness (InvSimpson) and diversity (Shannon) within the caeca samples showed values significantly higher in the caeca of birds reared on the conventional farm in comparison to the antibiotic-free farm. The bacteria biodiversity within the GI tract is considered an indicator of good health, and it was expected to be higher in the caeca of chickens not treated with Amoxicillin and Sulfadimethoxine/Trimethoprim. However, these antibiotics are only partially absorbed in the gut ,43, and  poultry  and is i poultry .In this pilot study we investigated for the first time the microbiome of the caeca of a bird and that of the corresponding carcass. The results showed that the caeca and carcasses of the same flock positively correlate one with the other. However, the correlation between the microbiome of the caeca and the carcass of the same bird was not stronger than that with other caeca and carcasses of the same flock. Therefore, the target analysis of caeca and carcass of the same animal does not provide any added value in comparison to the microbiome analysis at flock level. It is also clear from Besides the qualitative and quantitative differences in the most represented functional genes identified in the caeca and on carcasses from the animals reared in the conventional and antibiotic free farm, the most relevant result concerns the antibiotic resistant genes and the total antimicrobial resistance load. In relation to the ARGs, the multi antimicrobial extrusion protein (Na(+)/drug antiporter)-MATE family of MDR efflux pumps was significantly higher in the caeca of birds reared on the conventional farm in comparison to the antibiotic-free farm, along with few other ARGs. The MATE gene family is widely distributed in both Gram-positive and Gram-negative bacteria and contributes to the intrinsic, acquired, and phenotypic resistance of bacterial pathogens . MoreoveFor both caeca and carcasses, the overall antimicrobial resistance abundance per drug class did not show significative difference between the birds collected in the two tested farms, while specific differences were observed between drug classes associated to caeca and carcasses. The drug classes identified in both caeca and carcasses largely overlap with those identified by Munk  in the fGenes coding for resistance to vancomycin were identified among the ARGs with normalized mean values of abundance >1000 in at least one tested group. In accordance with other authors ,52, vancOverall, the results of this pilot study and the scientific literature demonstrate that each intervention in whatever processing step that the chicken and poultry meat is at, as with other food productions, is affected by the existing microbiome and resistome shifting and changing from farm to fork. Therefore, building robust, comparable, and representative databases of animal-, farm-, food- and production environment-associated microbiomes and resistome from farm to fork, as it is done for individual foodborne isolates and indicator microorganisms , would cAll in all, the results of this research indicate that post-harvest steps withdraw the positive effects of antibiotic-free rearing on carcass microbiomes. Therefore, it is crucial to assess the contribution of both transport and slaughter on carcass contamination and spreading of ARGs to identify possible mitigation options addressing consumer concerns regarding antimicrobial resistance and enhancing the positive impact of the European legislation, as well as the economic and management efforts of producers to rear antibiotic-free chickens.


This study reports the incorporation of small guest proteins by diffusion (soaking) into the solvent channels of a host protein crystal, visualized in time and space by microscopic techniques. The results represent a first step towards the realization of a protein-based crystal host  system that may be used in the future as a carrier/entrainment system for protein guests and/or for guest-protein structure determination. Escherichia coli tryptophan repressor protein (ds-TrpR) that are extensively domain-swapped and suitable for incorporating guest proteins by diffusion, as they are robust and have large solvent channels. Confocal fluorescence microscopy is used to follow the migration of cytochrome c and fluorophore-labeled calmodulin into the solvent channels of ds-TrpR crystals. The guest proteins become uniformly distributed in the crystal within weeks and enriched within the solvent channels. X-ray diffraction studies on host crystals with high concentrations of incorporated guests demonstrate that diffraction limits of ∼2.5 Å can still be achieved. Weak electron density is observed in the solvent channels, but the guest-protein structures could not be determined by conventional crystallographic methods. Additional approaches that increase the ordering of guests in the host crystal are discussed that may support protein structure determination using the hostal system in the future. This host system may also be useful for biotechnological applications where crystallographic order of the guest is not required.Soaking small molecules into the solvent channels of protein crystals is the most common method of obtaining crystalline complexes with ligands such as substrates or inhibitors. The solvent channels of some protein crystals are large enough to allow the incorporation of macromolecules, but soaking of protein guests into protein crystals has not been reported. Such protein host crystals  incorporating guest proteins may be useful for a wide range of applications in biotechnology, for example as cargo systems or for diffraction studies analogous to the crystal sponge method. The present study takes advantage of crystals of the  However, protein crystals that diffract X-rays are not always easy to obtain, partly because, unlike crystals of small molecules and compounds, they typically contain large amounts of solvent: between ∼25% and ∼75% of the total crystal volume  in an extended domain-swapped network represents a host with favorable properties, as described in this work. Horse heart cytochrome c  and fluorophore-labeled human calmodulin  are used as spectroscopically detectable guest proteins. Incorporation is monitored in time and space by conventional light microscopy and CLSM. This approach allowed the estimation of the volume fraction of the guest protein CaM in the solvent channels as a function of soaking time, indicating that very high guest contents are achievable to meet a basic requirement for structure determination. Diffraction analysis was used to compare the resolution of the TrpR crystals before and after soaking and to evaluate their applicability for guest-protein structure determination. The results show that TrpR is a useful host system for guest-protein entrainment; however, structural information about the guest proteins could not be extracted using conventional crystallographic methods, and several ways are suggested in which the system could be further developed to reach this goal.The present investigation aims to build on this body of work to develop a system that incorporates small to medium-sized proteins by soaking into a protein host crystal . The expression and purification of TrpR  are described in Supplementary Section S1.1. Crystallization was performed using the hanging-drop vapor-diffusion technique according to previously reported conditions, with a reservoir consisting of 27.5–35%(v/v) 2-propanol, 100 mM NaCl, 100 mM HEPES pH 7.5  with properties very similar to the wild type in the domain-swapped TrpR (ds-TrpR) crystal form was used throughout this work  was purchased from Sigma–Aldrich in the form of a lyophilized powder with a purity of >99% according to the supplier’s information.The cloning, expression and purification of Ser17Cys CaM allowing site-specific fluorophore labeling .Prior to adding the guest protein for soaking, 2–4-day-old crystals with a largest dimension of 30–100 µm were first transferred into 2–3 µl drops containing only reservoir solution for 1–2 days. In the initial soaking trials, staining of the crystals was followed using a bright-field microscope. Solid CytC (as purchased in lyophilized form) or Texas Red-labeled CaM (lyophilized) were added with a spatula to the crystal drop until no more solid material dissolved during addition  was prepared by mixing a solution of 300 mg ml−1 CytC in 100 mM HEPES, 100 mM NaCl pH 7.5 with reservoir solution in a 1:1 ratio. 2–4 µl of this CytC soaking solution was applied as a droplet on a new cover glass, into which TrpR host crystals of 20–50 µm were transferred. The crystals in the soaking solution were placed as hanging drops over 500 µl reservoir solution to prevent evaporation. Crystals were transferred to microscope slide wells with the soaking solution  just prior to imaging.For the imaging of CytC incorporation using CLSM, a CytC soaking solution  2-propanol, 50 mM NaCl, 50 mM HEPES pH 7.5. To follow soaking for the initial 40 min, crystals grown in 3–4 µl hanging drops were transferred using a crystal loop into a microscope slide well containing 4.5 µl Alexa532-CaM soaking solution. To follow soaking for longer times (>1 day), the crystals were transferred into new drops containing the Alexa532-CaM soaking solution and were kept as hanging drops over 500 µl reservoir solution to prevent evaporation. The crystals were transferred to microscope slide wells with the soaking solution just prior to imaging.For soaking studies with Alexa532-CaM, 30 µ−1, i.e. 14 mM, CaM with 0.2% Alexa532-CaM in 10 mM Tris–HCl pH 7.5) was mixed in a 1:1 ratio with reservoir solution to give a final soaking solution concentration of 110 mg ml−1 CaM with 0.1% Alexa532-CaM in 5 mM Tris–HCl, 15.6% 2-propanol, 50 mM NaCl, 50 mM HEPES pH 7.5. TrpR crystals in 3–4 µl hanging drops were first transferred with a crystal loop into a new drop containing 3–4 µl of this solution and, after seven or 15 days of soaking, were subsequently transferred to a microscope slide with the soaking solution. For imaging in the initial phase of soaking, TrpR crystals were transferred from the hanging drop into a well containing 4.5 µl soaking solution consisting of 88 mg ml−1 (5.5 mM) CaM with 0.1% Alexa532-CaM and were visualized after 5 min of soaking.For experiments with higher guest-protein concentrations and long soaking times (weeks), a protein stock solution of CaM  with reservoir solution, were applied to the wells and measured with CLSM using the same settings as for the later imaging of host crystals soaked with low concentrations of CaM. The final volume in the wells was 4.5 µl. The slides were mounted on a Leica SP5-X MP CLSM  and images with 355 nm excitation were taken using a 40× water-immersion objective . For imaging with 405 nm excitation an inverted Leica SP5 CLSM  was used, as this wavelength was not available using the SP5-X MP. Emission was recorded in 10 nm intervals as λ scans between 380 and 770 nm and between 415 and 755 nm, respectively.For CLSM imaging, TrpR host crystals or protein solutions were transferred into ten-well microscope slides with 30 µm depth (Thermo Fisher) and covered with a cover slip to avoid evaporation, thus allowing imaging over longer time periods of up to 15 days. Crystals were either transferred from the drop using 0.2–0.3 mm mesh LithoLoops (Molecular Dimensions) or by pipette (0.5–1 µl) into a well pre-filled with either reservoir solution (control) or the corresponding soaking solution (see Section 2.3Section S2.1). Crystals soaked with Alexa532-CaM or 110 mg ml−1 CaM with 0.1% Alexa532-CaM were imaged using an excitation wavelength of 528 nm and emission at 550–598 nm and the same CLSM settings as for all crystal imaging experiments. All images for different crystals and those used for quantification were recorded using identical settings for gain, pinhole, zoom and laser intensity as for the reference solutions of Alexa532-CaM. Images were analyzed using ImageJ within the Fiji software package and Leica Application Suite X (LASX 3.3.0).For Texas Red soaking, imaging was carried out with excitation at 594 nm and detection of fluorescence emission between 615 and 674 nm (Supplementary 2.5.−1 CaM (with 0.1% Alexa532-CaM) or 150 mg ml−1 CytC (from the same drops as used for CLSM) were cryocooled with liquid N2 after additional soaking for <1 min with 25% ethylene glycol as a cryoprotectant, which was also added to the respective CaM or CytC soaking solution used for crystal soaking. In data sets from the protein-soaked crystals, 4000 frames were collected with 0.1° rotation and a detector distance set to a diffraction limit of 2.0 Å at 0.97 Å wavelength. Processing was performed with XDS  using MOLREP  soaked for two weeks with 110 mg ml3.3.1.et al., 1985et al., 2004a) in a process akin to runaway domain swapping . The crystals meet the chemical definition of a gel . The channel size may thus be suitable to host small to medium-size guest proteins with a radius of gyration up to ∼5 nm, corresponding to a molecular mass of up to ∼50 kDa, depending on shape and dynamics  of 1.28 and 2.25 nm, respectively (summarized in Supplementary Table S1), sizes that in principle allow these proteins to enter the ds-TrpR solvent channels. Both proteins have suitable absorbance and fluorescence spectra. The heme group of CytC is a natural chromophore that is covalently attached to the protein via two thioether links to Cys14 and Cys18, and the Fe atom is coordinated by the protein side chains of His18 and Met80. Heme absorbance (red color) in the visible region can be used to monitor migration in TrpR crystals by bright-field microscopy, and its fluorescence can be used in confocal imaging. A cysteine-containing variant, Ser17Cys, of the otherwise cysteine-free CaM was used for specific covalent labeling with fluorescent Texas Red Maleimide or Alexa Fluor 532 (Alexa532) Maleimide dyes. Texas Red can be detected by its absorbance (blue color) in bright-field microscopy as well as by its red fluorescence at 615–674 nm with excitation at 594 nm, and Alexa Fluor 532 by its fluorescence at 550–598 nm with excitation at 532 nm.CytC (12 kDa) and CaM (17 kDa) have been extensively studied by X-ray crystallography and NMR, and their dimensions and the following specific properties suggest that they may be suitable as guests. Both proteins are highly soluble in aqueous buffers and could be dissolved in the crystallization solution used to form ds-TrpR at concentrations of ≫200 mg mlSupplementary Fig. S1. In brief, CytC or Texas Red-labeled CaM were added stepwise as lyophilized powders directly into droplets containing ds-TrpR crystals not older than seven days and of ∼50–300 µm in their longest dimension. Additions were continued until no more solid material went into solution within a few minutes. Staining of the ds-TrpR crystals was followed over time by bright-field microscopy, with a red or blue color detected after 1–2 days that appeared to extend throughout the crystals . The color of the guest proteins as judged in the bright-field microscope persists for days when the soaked crystals are transferred into new droplets without guest protein. Slight destaining is observed at the crystal edges, but the centers of the crystals remain colored for several weeks (data not shown). Crystals of ds-TrpR retained their macroscopic hexagonal bipyramidal shape throughout the staining, destaining, microscopy and diffraction analyses described below.The soaking procedure with CytC or CaM for bright-field microscopy as described below is shown in 3.3.Supplementary Table S2). The host crystals do not show background fluorescence with 528 nm excitation. The same CLSM settings were later used for imaging Alexa532-CaM-soaked crystals. In contrast, CLSM emission scans of host crystals with a 355 or 405 nm excitation wavelength show significant fluorescence background emission between 420 and 650 nm, with a peak at ∼465 nm . This background fluorescence is likely to be related to ds-TrpR in crystalline form, as a solution of dimeric TrpR at 4.2 mg ml−1 did not show detectable emission by CLSM with the same settings and shows no absorbance above 320 nm . The origin of this autofluorescence from ds-TrpR crystals was not determined. However, lysozyme crystals also are reported to exhibit autofluorescence of unclear origin when excited in the near-UV range . With excitation at 355 nm, the emission peak is red-shifted from ∼465 to ∼475 nm and a new peak is detected at 510 nm with ∼25% increased intensity at this wavelength for CytC-soaked compared with unsoaked crystals. With excitation at 405 nm, several spectral changes are observed close to the CytC heme Soret band absorption maximum at 410 nm: the emission peak is red-shifted from ∼490 to ∼505 nm and has fourfold to fivefold higher emission intensity, a new peak is detected at 550 nm  and the emission intensity between ∼500 and 650 nm is increased . Although CytC quantification is complicated by the background fluorescence of ds-TrpR crystals, confocal microscopy with 405 nm excitation was able to follow the penetration of CytC into the crystals semi-quantitatively over time and distance by taking advantage of the increase in fluorescence intensity above 600 nm, where host-crystal autofluorescence is nearly absent and CytC fluorescence can still be detected (Supplementary Table S2). Images with the focal plane through the center of crystals soaked with CytC for one day show more intense and homogeneous fluorescence intensity than the background fluorescence of nonsoaked host crystals, suggesting that the guest may be nearly uniformly distributed . Crystals soaked for shorter times, and some large crystals soaked for up to two days, show higher fluorescence at the crystal edges than in the center (data not shown), as also described also for CaM soaking. After less than one week of CytC soaking the fluorescence was typically homogeneous in all focal planes of the crystals, suggesting that the guest distributes evenly throughout the crystals.A closer comparison of emission spectra by CLSM reveals significant differences between host crystals with and without CytC guest protein when excited in the near-UV range  Alexa532-CaM and fluorescence was followed in the SP5-X microscope with excitation at 528 nm and emission around the maximum between ∼550 and 589 nm. Crystals soaked with the guest protein for a few minutes show Alexa532 fluorescence only in focal planes at or close to the surface . In contrast, the small-molecule dye Texas Red Maleimide alone shows rapid staining throughout the entire crystal on this timescale . After one day of soaking, Alexa532-CaM fluorescence is strongest at a depth of 10–15 µm from the surface of ∼40 µm crystals. After six days, confocal images show an even distribution of the guest throughout the crystals, with increased Alexa532-CaM fluorescence at the focal plane through the center  of the crystals compared with the images after one day of soaking .Host crystals of ∼20–∼50 µm in the longest dimension were soaked with 0.26 mg mlce Fig. 5a. In coSupplementary Fig. S6) was linear over the observed range of intensities. From this curve, the uniform fluorescence of the crystals after six days of soaking corresponds to ∼0.12 mg ml−1 (7 µM) Alexa532-CaM.Alexa532-CaM fluorescence in the crystal has the same spectrum as Alexa532-CaM in solution and was used to quantify the guest in the host crystal by assuming that the dependence of fluorescence intensity on concentration is the same in crystals as in solution. A standard curve for Alexa532-CaM in solution determined using the confocal microscope with the same settings . The maximum fluorescence intensity there is 95 arbitrary units (AU), compared with 45 AU for the surrounding CaM solution and zero or negligible fluorescence at the center of the crystal. After one week of soaking with 110 mg ml−1 CaM containing 0.1% Alexa532-CaM, a host crystal of ∼40 µm in the longest dimension shows increased fluorescence throughout . The fluorescence within 5–10 µm of the crystal surface  exceeds the linear response range of the standard curve under the settings used. The center of the crystal has greatly increased fluorescence compared with the soaking solution, with a minimum fluorescence intensity of 200 AU, which is also above the linear range of the standard curve. After two weeks of soaking, the Alexa532-CaM fluorescence exceeds the linear response range throughout the entire inside of the crystal . This result indicates that the host crystals become enriched in CaM relative to the surrounding CaM solution.To permit the structure determination of a guest in the channel, it is expected that higher concentrations of the guest proteins are needed than those used in the initial soaking experiments. Using only labeled CaM would bring the fluorescence intensity above the linear range of detection for the microscope under the settings used. Therefore, concentrated solutions of unlabeled CaM were supplemented with ∼0.1% Alexa532-CaM to allow the CaM distribution in the crystals to be followed by CLSM using the same settings for all images of the time series described below. The fluorescence and the corresponding plot of intensity from a focal plane through the center of a crystal imaged 5 min after transfer into 88 mg mlal Fig. 5d. The mut Fig. 5d. The fal Fig. 5d. This 3.4.guest describes the fraction of the volume (Vguest) of the crystal occupied by the guest protein, Vcrystal = Vhost + Vsolvent + Vguest.For the hostal system to be developed in the future towards crystallographic structure determination of protein guests, the guest-protein content must represent a sufficient fraction of the solvent-channel volume  plus the volumes of solvent and guest. The guest content [in %(v/v)] within the host crystal derives from Φguest {the guest content [%(v/v)] equals 100 × Φguest}. The volume fraction of the guest protein in the crystal, Φguest, is obtained from the experimentally estimated mass concentration of the guest protein in the crystal and the density of the guest protein (ρguest):guest, from which the guest content in the crystal can be determined assuming a homogeneous distribution of the estimated protein concentration throughout the crystal solvent. The theoretically and experimentally determined densities of proteins range from ∼1.22 to ∼1.50 g cm−3 depending on the molecular weight. For proteins of molecular weight <20 kDa the experimental density is generally close to the value of 1.37 g cm−3  can be determined by dividing the estimated concentration in mg ml−1 measured in the crystal by the average protein density of 1.37 × 103 mg ml−1 and correcting for the solvent fraction, 75%. Fig. 6guest in the crystal solvent using equation (2)The total volume of the crystal, −1 guest protein in the host, corresponding to a guest content of only <0.01%. As described above, high Alexa532-CaM concentrations during soaking resulted in guest-protein enrichment in the channels compared with the concentration in the soaking solution, with the guest concentration estimated to be at least 400 mg ml−1 after two weeks of soaking . This concentration corresponds to a guest content in the crystals of ∼30% . Although there are many possible sources of error in this estimate, its order of magnitude is similar to the TrpR content in the crystals of 25% (volume fraction 0.25), suggesting that long soaking times with high concentrations of the guest achieve guest contents that may support crystallographic structure determination if the guests are sufficiently ordered in the crystal.Soaking with low concentrations of Alexa532-CaM results in 0.12 mg mlFurthermore, crystal staining with CytC followed by bright-field microscopy, and its fluorescence spectral shift in CLSM indicating uptake, suggest that CytC also enters host crystals, although following the CytC guest-protein distribution quantitatively over time by CLSM and estimation of its content were challenging. The slightly smaller radius of gyration and the larger diffusion coefficient of CytC suggest that its migration in the crystals should be at least as facile as that of CaM, although the unknown factors leading to enrichment of CaM in the crystals may differ for CytC.3.5.−1 CaM (with an estimated guest concentration of 400 mg ml−1 in the channels as described above) or 130 mg ml−1 CytC. In each case crystals were from the same drops as used for CLSM. Crystals were tested by X-ray diffraction on the BioMAX beamline at MAX IV, Lund. Reference diffraction data for nonsoaked host crystals alone, set up for growth at the same time as the crystals for soaking, were measured during the same experiment. All data sets were processed in P6122 and also in P1 . The choice to additionally carry out structure determination in P1 was made in order to consider the additional possibility that the guests may follow the same lattice but not the same space-group symmetry as the host. Data sets were collected for four individual CaM- and CytC-soaked crystals with maximal resolutions of 2.45–2.90 Å and 2.75–2.95 Å, respectively (cutoff according to the shell with CC1/2 > 0.5 in the first XDS run in either P6122 or P1). The resolution of the soaked crystals is only slightly decreased  compared with the test set of four nonsoaked ds-TrpR host crystals measured in the same experiment . The unit-cell parameters show no large differences among the data sets, but Rmeas for merging in P6122 is higher, especially for the CytC-soaked crystals (>15%) compared with nonsoaked ds-TrpR crystals (∼12%) (Supplementary Table S6). These differences in the merging statistics may directly or indirectly be a result of the soaking procedure and may be caused by, for example, crystal deterioration or contributions to the diffraction by a guest that may not follow the host symmetry perfectly. However, similarly as for merging in P6122, merging in P1 also results in an elevated Rmeas for the soaked crystals . Analysis by phenix.xtriage in the Phenix suite  as a search model. The host structure solved in P1 does not show any obvious differences compared with the P6122 structure, showing that a breakdown in symmetry is unlikely to be the origin of the higher merging statistics described above. Refinement statistics are summarized in Table 1Supplementary Tables S7–S9). The Rfree/Rwork values are slightly elevated for the guest-soaked compared with unsoaked ds-TrpR crystals. The elevated values may reflect several factors, including differences in resolution or some contribution from the guest. However, the effect is over the full resolution range and is not the result of badly modeled low-resolution data (Supplementary Table S7), and thus does not seem to be caused by erroneous bulk-solvent correction in the guest-soaked crystals.The structures of ds-TrpR from the best-diffracting crystals from each soaking experiment and from nonsoaked crystals were determined by molecular replacement in Section S2.3). Weak electron density detected in the solvent channels could not be identified as partial or complete structures of CaM or CytC in the corresponding soaked crystals. To visualize this weak density, the electron density is shown in Fig. 7Fo − Fc maps at high noise levels (0.6 r.m.s.d.) and as blurred maps to enhance low-resolution information. Blurring of the 2Fo − Fc maps was used in contrast to map sharpening, which is typically performed to increase the quality of high-resolution information in crystal structures , but not for the nonsoaked ds-TrpR host, features of connected electron density are visible when map blurring is applied. The observation of additional density is consistent with what is seen in comparison of maps on the absolute scale of electrons per unit volume . Inspection of maps after refinement without bulk-solvent correction indicates that the channel density for guest-soaked crystals is enhanced compared with nonsoaked crystals , suggesting that bulk-solvent correction may contribute to suppressing the guest density. The additional electron density observed in CaM-soaked crystals cannot be explained by the different resolutions of the data sets, as the same map features are seen when all of the data are truncated to the same (lower) resolution of 4 Å . Although these results suggest that the channel density is due to a scattering contribution from the guest protein, it is not clear at present how useful it may be for future structural interpretation.Molecular-replacement searches readily identified the ds-TrpR host in crystals soaked with CaM or CytC, but no solution could be found for the guests when their structures were included as search models in molecular replacement  for soaking results in an apparently at least fourfold higher guest-protein concentration in the crystal compared with the soaking solution, suggesting an enrichment effect of the guest in the mesoporous crystals of the host. Enrichment of proteins and peptides in silica-based mesoporous materials has previously been reported  colored guest proteins observed by bright-field microscopy was a first indication that proteins of this size can enter the crystals, but did not allow conclusions about their penetration depth or uniformity of distribution. As indicated by the work of Cvetkovic  al. 2004, CLSM iset al., 2006et al., 2017et al., 2000et al., 2014et al., 2000et al., 2004et al., 2006It will be useful to better understand and develop models for the migration of proteins through a protein-based mesoporous system such as the ds-TrpR crystals used here. Unconstrained diffusion times for single protein molecules based on Brownian motion , and ds-TrpR crystals do not develop crystal pathologies such as twinning upon soaking. Despite the very high solvent content of ds-TrpR crystals, they are shown here to be mechanically stable and to tolerate these changes in the environment without physical breakage or significant loss of diffraction quality. These observations from the soaking studies show that the crystals can tolerate at least some chemical changes in the surrounding solution. Future work may therefore explore whether host–guest interactions can be promoted by modification of the crystal chemical environment, for example, by manipulation of the Hofmeister series of ions that may favor or disfavor electrostatic or hydrophobic interactions.4.3.122 symmetry of ds-TrpR with a rough estimate of ≥37% solvent content at maximal guest content, although they need not fully or partially follow the 6122 symmetry of the ds-TrpR host. Guests of up to ∼50 kDa could in principle be accommodated in the same primitive lattice as the host but without internal symmetry (P1). The larger the guest, the higher the likelihood that the guest will not follow the host symmetry because there are fewer possible ways to occupy the limited space, which also depends on the guest shape. In the diffraction data sets analyzed here there was no indication that the guests are ordered but do not follow the host symmetry or occupy a lattice different from that of the host.Guest proteins of up to ∼36 kDa could theoretically follow the hexagonal symmetry of ds-TrpR crystals, as estimated very roughly . This is an extreme upper limit, however, considering that 0% solvent content is not realistic and that the pore shape also contributes to the space available for the guest. Thus, guest proteins significantly smaller than 35 kDa are most likely to meet the requirements of size and symmetry for the ds-TrpR hostal system. As the guest proteins studied here, CytC (12 kDa) and CaM (17 kDa), are well below this limit, they are theoretically suited to penetrate the channels and follow the 64.4.et al.  and a nearly equal molar ratio of CaM guest to TrpR host (1.1:1). Such enrichment over the surrounding bulk may be due to favorable interactions between CaM and the host channels or due to a net gain in entropy if calmodulin replaces a significant number of ordered water molecules from the channels. High guest content within the host crystals is a necessary, but not sufficient, requirement to achieve the high site occupancies needed for conventional structure determination of the guests. If all guest molecules follow the host crystallographic symmetry and have limited intrinsic flexibility or internal disorder, the concentrations achieved here with CaM are expected to yield occupancies that are sufficient for structure determination. However, structural information on the guests was not easily obtained by conventional structure-determination methods. This observation suggests that the guest has a large degree of disorder in the host channels, making the occupancy at individual crystallographic sites too low to solve the structure in conventional ways; positional disorder and/or internal guest dynamics may be responsible.The present work shows that starting from bulk concentrations of ∼100 mg mll-tryptophan (l-Trp). l-Trp binds to ds-TrpR in a position equivalent to its binding site in dimeric TrpR , a result that is not necessarily expected because the residues of the binding site arise from three polypeptide chains in ds-TrpR rather than two in native dimeric TrpR. This approach might be especially useful with smaller guests (for example peptides) that may be able to diffuse past the first tightly bound molecules that limit the space in the channels.Thus, the eventual success of a hostal method for structure determination is likely to depend on improving the order of the guest molecules experimentally so as to limit the number of guest conformations and improve the occupancy at crystallographic sites. The ds-TrpR host offers several approaches to increase the order of guest proteins by promoting specific and strong host–guest interactions while allowing adequate migration of the guest through the solvent channels. One potential way to order guests involves chemical tagging with mimics of the physiological ligand et al., 2004et al., in preparation) also reveal that TrpR bearing an N-terminal extension crystallizes in a domain-swapped form without degradation of diffraction quality and that the extension points into the solvent channels. This result suggests that engineering the host to include an N-terminal peptide representing a binding site as ‘bait’ for a peptide-binding guest protein such as CaM might enable targeting to the channels, enhancing crystallographic site occupancy that follows the symmetry of the host. In this context, the physiological interactions of CaM might be exploited as a proof of concept, as the binding of CaM or its individual domains to peptide targets is additionally dependent on calcium ions that can be used to trigger binding (Babu et al., 1985An additional feature of ds-TrpR crystals that may facilitate the ordering of guest proteins is that the N-terminus of the protein faces the crystal solvent channels (Lawson PanDDA maps (Pearce, Bradley et al., 2017et al., 2017B factors may be achievable in the future using the anchoring strategies suggested above, thus potentially making structure determination possible with careful density analysis. Diffraction methods that make use of crystal disorder may also be advantageous. It has, for example, been shown that taking the diffuse scattering signal from a diffraction experiment into account improves structure interpretation for less ordered parts of crystal structures (Chapman et al., 2017Recent advances in the fields of X-ray diffraction and the interpretation of low-level electron densities may additionally help to solve guest-protein structures in the future. The lowest limit of site occupancy for a molecule to be structurally elucidated using conventional crystallography is not completely clear, but it has been suggested (Deller & Rupp, 20154.5.et al., 2009et al., 2015et al., 2009et al., 2017et al., 2019et al., 2019In addition to using ds-TrpR as a hostal system for solving the crystal structures of guest proteins, the robust crystals of ds-TrpR, and similar hostals that may be developed in the future, may also be applicable in other fields of biotechnology. Protein crystals, especially of enzymes, have gained interest, for example, for use as ‘micro-bioreactors’ that serve to concentrate reactants in confined volumes, in line with other protein-encapsulation methods (Minten 10.1107/S2059798321001078/rr5201sup1.pdfSupplementary materials and methods, supplementary results, Supplementary Tables and Supplementary Figures. DOI:


Purpose: Debridement, antibiotics, and implant retention (DAIR) is an established treatment modality in periprosthetic joint infections (PJIs), but success rates vary. This study compared the success of DAIR for PJIs after a total hip arthroplasty (THA), with or without local antibiotic delivery with CaSOMethods: A retrospective review of DAIR for PJIs after THA performed between 2010 and 2018, including 41 patients is conducted. A total of 27 patients were treated by DAIR with local antibiotics with CaSOResults: Considering any reoperation as an outcome, 11 of 14 cases treated without AB-CaSOConclusion: The addition of local antibiotics with CaSO PJI is associated with high morbidity and mortality  and represents a high economic and logistic burden on the healthcare system .A two-stage exchange remains the standard therapy for PJIs . While being very successful regarding the eradication of infection, this option is associated with functional outcomes that are worse , with higher complications and increased mortality rates compared to implant-retaining procedures and one-stage exchange . PJIs may also be treated successfully by debridement, antibiotics, and implant retention (DAIR) if the implant is well-fixated and if biofilm-active antibiotic treatment is available . The reported success rates of DAIR, regarding the eradication of infection, vary from 35 % to 88 % . The variability in the results may be explained by the heterogeneity of the cohorts, the influence of the duration of the symptoms and of the causative microorganisms, variable definitions of success, and differing follow-up periods. Even a hard outcome, such as reoperation, may result in differences in analysis, as the persistence of infection is not necessarily diagnosed or reoperated and a suppressive antibiotic treatment is possibly started instead . Repeated DAIR procedures may also be successful, with persistence of infection after a first DAIR not necessarily requiring component removal . Nevertheless, the functional outcomes of successful DAIR correspond to results obtained after primary THA or TKA without PJI , which are much better than the functional outcomes after two-stage exchange .The efficacy of the antibiotic treatment, and thus the success rate of DAIR,may potentially be increased with local antibiotic delivery. Calciumsulfate  and reviewing the prospectively collected database of patients receiving local antibiotic treatment with CaSO®; Wright Medical Technology, Inc., Arlington, Tennessee) was introduced in our institution in 2015 and became routinely applied from 2017 onwards. The technical details regarding preparation of the beads are described elsewhere . Routinely, three 25 mL packs of Osteoset® loaded with 2 g vancomycin each were implanted into the hip joint after debridement. This quantity was usually reduced to one or two packs in case of severe renal failure, due to potential for hypercalcaemia. As ceftriaxone causes a major volume expansion, the number of packs, consequently, also had to be reduced as adaptation.The diagnosis of PJIs is based on current international guidelines . At least one of the two attending physicians of the hip team were involved in all cases, with one physician being replaced in 2015. Since 2010, an anterior approach, performed in supine position on a traction hemi-table applied to the operated leg, has been the standard in-house procedure for primary THA. Pre-existing, other approaches were also used, which was particularly appropriate for external cases referred for treatment. Debridement repeated within 72 h following the initial DAIR procedure was supposed to be second-look operations as part of the initial treatment plan and was not counted as separate DAIR procedure. While there was no strict rule to perform second-look operations, this was common practice in the early phase of the study. Modular components were always exchanged in DAIR procedures  but only once in the case of second-look operations. In case a second look was performed, only the first operation was considered for the Kaplan–Meier analysis. Postoperative antibiotic treatment followed international guidelines, including a biofilm-active drug administered as soon as possible and for a total duration of 12 weeks . Treatment in both groups was otherwise identical, except for the local application of antibiotic-loaded calcium sulfate  and any revision with implant exchange  as endpoints. The subgroup analysis stratified by treatment with and without CaSOPrior to this study, written general consent for such an analysis was obtained from all patients as a matter of routine. As this study only involved a retrospective review of anonymized data without any interventionfor study purposes, Swiss law requires no specific approval by an externalethical committee.3Among the 41 patients treated using the DAIR procedure for PJI after THA withstandard implants used for primary operations, 14 were treated between 2010and 2017 without local application of antibiotics, and 27 were treatedbetween 2015 and 2018 with AB-CaSOConsidering any reoperation as outcome parameter, 11 of 14 cases treatedwithout AB-CaSO4In this study, infection-free survival and the success rates of DAIR forhip PJI greatly improved when AB-CaSO®  reported in a study with low success rates in DAIR for PJI after THA , which is roughly equivalent to 25 mL Osteoset®, may also lead to a more favorable elution due to a bulk effect of the carrier material. Elution kinetics may also differ due to differences in the concentration of vancomycin and of tobramycin and explain the differences in the results .Our observations are in contrast to previous publications, which did notidentify the advantages for the local delivery of antibiotics in DAIR . This may be due to a variety of reasons. While PMMA remains the standard carrier for local antibiotic delivery, drug elution kinetics are mostly unfavorable . Aminoglycosides with bone cement serving as the carrier material may even be associated with worse outcomes in DAIR ; this is not only due to the requirement ofreoperation to remove the non-resorbable carrier . The choice of applied antibiotics may be another explanation. Aminoglycosides(gentamicin and tobramycin) are small molecules with a high solubility. When not covalently bound, these drugs rapidly elute from any carrier material, with the consecutive potential for systemic toxicity . In addition, the mode of action of the aminoglycosides may be inadequate for orthopedic indications, as intracellular penetration that is necessary for the primary mode of action of inhibition of the protein synthesis is an oxygen-dependent active transport which is reduced in an acidic environment . In this case series, aminoglycosides were only added in two cases (Table 1) and with the intention of optimizing the effect of vancomycin or ceftriaxone, which are both known to have favorable elution kinetics from CaSOThe current study has several limitations which have to be discussed.Patient care followed standard guidelines regarding patient selection andsystemic antibiotic treatment . A rather small team of attending surgeons and infectious diseases specialists was involved, which ensured a certain standardization and continuity. Whereas the principles of the DAIR procedure were applied during the whole study period, the quality of the procedure may vary. The inclusion period was set to begin in 2010, as this corresponds to a switch in our department to the anterior approach as the standard approach in primary THA. However, CaSOWhile corresponding to the spectrum of bacteria usually encountered in hipPJIs , the microbiology in this case series was heterogeneous (Table 2). It is well known that the outcome for certain microorganisms is worse than for others . Nevertheless, the spectrum of microorganisms may not explain the differences in results (Table 2). Failure happened nearly exclusively within the first few months after DAIR. This is in accordance with results from other studies investigating virulent species like streptococci and methicillin-resistant staphylococci . Extending antibiotic coverage to gram-negative bacteria was only seldom necessary (Table 2). Standard systemictreatment always was 12 weeks, following the regimes usually recommended. Antibiotic treatment was not extended, as recommended recently for streptococci . No patients included in this study had long-term suppressive antibiotic treatment.Curing infection is only one relevant issue among other parameters.Improving outcomes of DAIR procedures is very important, as functionaloutcomes for successful DAIR are much better than after exchange procedures are performed, particularly if staged . Reported success rates of DAIR, regarding the eradication of infection, vary from 35 % to 88 % . In the group without local antibiotic application, the mid-term success rate was comparatively low, which points, of course, to other issues. On the other hand, the success of DAIR with AB-CaSOIn conclusion, this study shows a major advantage for local antibiotictreatment with CaSO