Background: Antimicrobial resistance (AMR) is a global threat and the antimicrobial stewardship program (ASP) is a globally used tool to combat AMR. There is little information on the views among Pakistani physicians regarding AMR and the benefits of hospital antimicrobial stewardship implementation. This study was designed to explore the physicians’ views about ASP. Methods: Qualitative face-to-face and telephonic interviews were conducted by using purposive sampling method with 22 physicians working in seven tertiary care public hospitals of Punjab, Pakistan. All interviews were audio recorded and transcribed verbatim. Qualitative software was used, and a thematic analysis was conducted. Results: Three broad themes were identified: (1) the growing concern of antimicrobial resistance in Pakistan, (2) the role(s) of healthcare professionals in antibiotic prescribing, and (3) managing antibiotic resistance in hospitals. Inadequate resources, poor healthcare facilities, and insufficiently trained medical staff were the major hurdles in ASP implementation in Pakistan. Conclusions: Our study found a poor familiarity of hospital ASP among physicians working in public sector tertiary care teaching hospitals, and a number of distinct themes emerged during this study that could be helpful in establishing the concept of hospital ASP in Pakistan. Overall, physicians showed a positive attitude towards the enforcement of ASP in all healthcare settings, including teaching hospitals. Antimicrobial resistance (AMR) is a growing global challenge . It is eBeing a global issue, AMR is also increasing in Pakistan at an alarming rate. The major reasons of antibiotic resistance include unjustified use of antibiotics , sale ofThe implementation of an antimicrobial stewardship program (ASP) in both hospital and community settings is one of the key solutions to addressing the challenge of AMR ,17,18,19In Pakistan, efforts to combat AMR were initiated by the Medical Microbiology and Infectious Diseases Society of Pakistan (MMIDSP) in 2014 in order to develop and implement indigenous ASPs throughout the country . UnfortuThis was an explorative and qualitative study design that was based on face-to-face and telephonic in-depth interviews with physicians to determine their views about ASP. Semi-structured interviews are useful when collecting attitudinal information or when the research is exploratory. This design has several advantages, including its flexibility and ability to provide an in-depth investigation of the attitudes, experiences, and intentions of participants . FurtherThe study was conducted in three cities  of Punjab, which is the most populous province of Pakistan . These cA semi-structured interview guide was developed after a thorough literature survey ,44,45,46Participants included physicians who prescribe antimicrobials, were purposively sampled from seven tertiary care public teaching hospitals. A snowball sampling strategy to recruit participants was used . EnrolleAll interviews were conducted in English, since physicians were fluent in English . The duration of an interview ranged between 30 to 45 min. Refreshments were provided, but no other incentives were given for participation. Participation was voluntary, and all the participants were informed about their right to withdraw from study at any time. The identities of the participants were kept confidential from hospital employers and supervisors. In this way, participants had the opportunity to freely express their views. Physicians were included in this study only if they dealt with patients with infectious diseases and worked in public sector tertiary hospitals. Physicians working in private hospitals were excluded.All interviews were audio-recorded, transcribed verbatim, and de-identified. The major themes and sub-themes were identified by the research team after thematic content analysis by utilizing steps previously described elsewhere ,49,50. TEthics approval was obtained from Biomedical Ethics Committee of Xi’an Jiaotong University (no. 2018-45). The study was also approved by the University of Veterinary and Animal Sciences, Lahore, Pakistan. Written and verbal consent was taken from all enrolled participants prior to the interviews. In total, 22 interviews were conducted. Seventeen participants were male. Most (59.09%) of the participants were between 20–40 years of age. Seven participants were split between 16–20 years of practice .The themes that emerged from the semi-structured interviews were broadly classified into three major categories: (1) growing concern of antimicrobial resistance in Pakistan, (2) the role of healthcare professionals in antibiotic prescribing, and (3) managing antibiotic resistance see .“Resistance of course is increasing in Pakistan and, unfortunately, we are facing this problem with higher intensity every year (...) Now, at this stage I feel that a simple sore throat infection that was easily cured by erythromycin is no longer cured due to the emergence of resistance.” (P7)“There is no doubt that there is a strong relationship between irrational use of antibiotics with antibiotic resistance and it is continuously growing in Pakistan.” (P2)Questions related to AMR were asked of all the participants. Most of them were of the view that antibiotic resistance is escalating gradually in Pakistan and rational approaches are urgently needed to help tackle it.The reasons of antibiotic resistance were well explained by the participants. “Unfortunately, there are (a) number of other so-called doctors such as unlicensed practitioners who most of the times prescribe antibiotics in an irrational way, thus increasing the risk of antibiotic resistance.” (P6)“In our country, unlicensed practitioners prescribe antibiotics without any rational approach and (are) amplifying the problem of antibiotic resistance.” (P16)The participants revealed the presence of unlicensed practitioners as a major problem towards the increasing rate of antibiotic resistance due to their unjustified medical practice.“There is a lack of appropriate diagnostic facilities, especially in basic health units and district hospitals. This further promotes the use of antibiotics without culture sensitivity tests.” (P8)“Physicians in the periphery and remote areas mostly use broad spectrum antibiotics and they do not bother to recommend lab tests, maybe due to (the) absence of diagnostic facilities.” (P5)Some physicians said that the absence of standard diagnostic facilities contribute toward the irrational prescribing of antibiotics.“Non-compliance with antibiotic treatment is common among patients, as most of them don’t complete their treatment course and when they use the same medicine in future, it is unable to cure them due to the emergence of resistance.” (P9)“They (patients) will stop their antibiotics once they think that they are feeling better and do not complete the recommended duration of therapy as we have instructed them.” (P12)“Resistance against antibiotics is increasing just like (in) other countries (….) there is a need to educate and encourage patients to complete their antibiotic course to prevent the risk of resistance against antibiotics.” (P21)The participants suggested the need of adequate patient education about antibiotic use and ways to reduce or prevent antibiotic resistance.“Unfortunately, self-medication is very common, and you can take any drug like antibiotics freely from the medical store or pharmacy without a prescription.” (P5)“In my view, the origin of resistance is the inappropriate use of antibiotics by the community. This risk will be higher if they are able to get these pills without any prescription.” (P11)“I think the government should take meaningful steps to prevent the sale of antibiotics without a prescription.” (15)The participants recommended the enforcement of strict laws to limit the purchase of antibiotics without a prescription in order to cut the risk of antibiotic resistance.“A major weakness in government sector hospitals is (the) lack of mutual cooperation among different healthcare professionals, which could affect the rational use of antibiotics in hospitals.” (P3)“Antibiotic prescribing is a team (effort) in which every professional is doing his/her job (……) being a physician, we can discuss about antibiotic therapy with pharmacists and nurses. Obviously, microbiologist will be helpful for lab results. So, in this way, we will have evidence-based antibiotic therapy, which will improve the quality of life of patients, but unfortunately this coordination is missing.” (P13)“No doubt if all the stakeholders of our healthcare system including physicians, pharmacists, nurses, and microbiologists work in coordination then it will definitely improve antibiotic usage within a hospital.” (P15)Participants highlighted the need for coordination among different healthcare professionals, including physicians, nurses, microbiologists, and pharmacists, to help improve antibiotic prescribing and use in hospital patients.“[Patients] sometimes demand antibiotics and try to influence our clinical judgment, especially in government hospitals. They know basic information about antibiotics, like names of certain very common antibiotics, which (were) possibly tracked from friends or the Internet, and they are pre-occupied with receiving a specific antibiotic. So, they even ask us to prescribe that antibiotic.” (P3)“This is a demand of patients to be given an antibiotic, but we try to prescribe only if it is suitable for them. Some patients come after evaluation from several doctors and show us their previous prescriptions and tell us which antibiotic showed better results, then in such situations, we sometimes prescribe the same antibiotic.” (P6)Some physicians also explained that patients tried to influence them by demanding antibiotics as part of their drug therapy despite the nature of their infection.“Well, there are certain infections like flu in which antibiotics should not be given. I try to counsel such patients and tell them that it’s nothing but an extra expense from your pocket. Most of the times, patients agree to what I say.” (P1)“We educate patients suffering from viral infections by saying that symptomatic relief is all that you need, and antibiotics use will be ineffective in this case. In reality, self-medication with antibiotics is very common in our community and the rate of compliance is higher if you prescribe a greater number of pills to the patients regardless of their clinical needs.” (P7)Participants were asked about how they educate patients if they ask for antibiotics for their viral infections.“Yes, we do have an infection control committee in our hospital, but at the moment I am not satisfied with its current performance due to [the] lack of its active role in reducing the risk of hospital-acquired infections as well in the development of infection prevention policies and treatment guidelines. Its performance must be improved to address resistance issues.” (P20)“The infection control committee exists in our hospital, but it is in a very pathetic condition. Its working is not up to the mark, probably due to insufficient funds and inadequate staff. (……) There is a need to improve its role to reduce antibiotic resistance.” (P3)Most of the participants were not happy with the role of infection control committees in their hospitals and suggested that its role should be enhanced to curtail antibiotic resistance.“To be honest, I am not aware of this program, but now I have some basic information about it after a discussion with you. It seems to be a new concept to rationalize the use of antimicrobials including antibiotics.” (P14)“Unfortunately, I do not have any prior information about this program.” (P13)During the interview, participants were asked about their understanding of hospital ASPs. Only a handful of physicians had some knowledge about an ASP. Detailed written information was provided to all participants to elicit their views on hospital ASPs.“I personally do not have any reservation in (regard to) its (ASP) implementation, as it is not only useful for patients but also for physicians.” (P11)“It is a very useful program and, being a physician, I have (a) very positive attitude towards ASP due to its benefits in (terms of reducing) antibiotic resistance, which will speed up the prognosis rate among patients. If we consider ASP in ideal condition(s) then, of course, this system will have multiple merits.” (P9)All participants interviewed showed a positive attitude towards implementation of ASP in Pakistani hospitals.“There will be a lot of barriers. For example, we have heavy patient loads and it would be too difficult for us to get a prior approval of an antibiotic from an ID physician every time (…) Secondly, there will be a need of huge financing to implement hospital ASPs.” (P8)“I perceive that budgeting, which is the main hindrance, and inadequate resources could be the major barriers in its implementation.” (P1)“We may not have enough qualified and trained professionals like ID physicians, nurses, microbiologists, and clinical pharmacists who have experience of working in (an) ASP healthcare facility.” (P17)Participants indicated several barriers in ASP implementation in Pakistani hospitals, but all those barriers could be overcome if government provides necessary support, including financing.“As per my opinion, the second one [formulary restriction] will be more effective, as this will restrict the use of antimicrobials. This restriction should be specific to every hospital, but some physicians may resist in order to seek approval before prescribing antibiotics.” (P7)“Prior approval of prescribing antibiotics will be more beneficial in Pakistan, as this will act as a check point and limit the prescribing of antibiotics by physicians, but this may also challenge the authority of the physicians.” (P4)“I think we should try to implement a combination of different ASP strategies to determine the best suitable and useful option.” (P12)There are different strategies that can be used to adopt ASPs in hospitals, such as prior approval from ID physician/formulary restriction, and prospective audit with feedback. There were mixed views from participants when the question related to a preferred strategy for hospital ASP implementation was asked.“Well, we must start this program from tertiary healthcare units and district hospitals, where there are more trained staff and experts. Once they understand it, then they will teach to their juniors and (knowledge will) trickle down from top to bottom.” (P1)“I think ASP should be implemented first in tertiary hospitals and then step by step to other hospital settings, including secondary and primary.” (P10)In Pakistan, there are different types of hospital settings, including tertiary care hospitals and secondary care hospitals. During the interviews, participants were asked about the type of hospital settings where ASP should be preferably implemented.“Yes, obviously, training is needed in order to get better understanding in the form of workshops or conferences and I think healthcare professionals should have (a) certification from an approved course in order to be the part of ASP.” (P5)“It will be a good option to train the physicians before the implementation of this program. We need to do some pilot project first and if results are positive then we can continue it with zeal.” (P10)“Well, training is always necessary in order to get better results. So, all healthcare professionals should be trained before the implementation of ASP in hospitals. In my view, the government should plan a training program accompanied with a certificate, so that only a trained healthcare professional could join the ASP team.” (P18)All participating considered ASP training as an important step before its adoption.The major themes of this study are antibiotic resistance as a problem in Pakistan, the role of healthcare professionals in antibiotic prescribing, and managing antibiotic resistance by the implementation of hospital ASPs in Pakistan. The present study has highlighted good familiarity among most of the physicians towards the antibiotic resistance problem in Pakistan. They were aware that this problem is gaining momentum and that rigorous efforts are needed to cope with this issue. The participants revealed some major factors for the prevalence of resistant microbes in Pakistan, including the sale of antibiotics without a prescription, as well as the presences of unlicensed medical practitioners, self-medication, and poor diagnostic facilities. These findings are similar to those in prior published studies ,53,54,55In this study, most of the participants were not satisfied with the performance of their infection control committee. So, there appears to be a need to strengthen the functions of infection control committees in hospitals, as this will help in the prevention of the nosocomial spread of infections and resistant pathogens. In the absence of control measures, hospitals may act as a reservoir of antibiotic resistant strains that could be disseminated to the community ,11,57. ASP is still a new approach in Pakistan to curtail the problem of antibiotic resistance. There has already been a lot of work reported in different developing countries ,58,59, bAccording to the results of this study, an ASP-certified training program would be of great benefit not only to physicians but also for nurses, microbiologists, and clinical pharmacists. All the participants agreed that training programs would improve their familiarity with objectives and core elements of ASP. Our study also found that a lack of resources in terms of both staff and infrastructure creates the major hurdles toward ASP implementation. Unfortunately, there are very few infectious disease physicians working in Pakistan, and the doctor to patient ratio is alarmingly low (1:1300) ,62. Two approaches for hospital-based ASPs are currently endorsed by national organizations and include preauthorization/formulary restriction and prospective audit with feedback ,64. The To the authors’ knowledge, this study is the first of its kind to investigate the views among Pakistani physicians regarding hospital ASPs. However, there are limitations that should be noted in this study. First, this study cannot depict a complete picture of all physicians of Pakistan, as it was conducted in only three cities of Punjab province. Having said this, considering that it took place in the most populous province of Pakistan and among experienced physicians working in different tertiary hospitals, the findings may be applicable to other parts of the country. Second, there is a potential researcher bias. To reduce individual biases, we involved several researchers with pharmacy backgrounds and knowledge of ASP concepts to assist with coding and theme development. Finally, only physicians from public sector hospitals were included, and views among physicians working in private hospitals may differ from those expressed in this study. In conclusion, a diminished understanding of hospital ASPs was found among physicians working in public sector tertiary care hospitals of Punjab, Pakistan. However, they have shown a positive attitude towards its enforcement in all healthcare settings, including teaching hospitals. Insufficient resources, poor healthcare facilities, and inadequate training of medical staff are the major hurdles in ASP implementation in Pakistan. The onus may now be on government policy to help initiate ASP in all public sector hospitals in the country.


Staphylococcus epidermidis at different stages of processing. The protocol was highly efficient at removing bioburden introduced at any stage of processing, with antibiotic treatment and drying the most efficacious steps. The antibacterial activity of non-antibiotic treated AM compared to VDAM was evaluated using minimum inhibitory/biocidal concentrations (MIC/MBC), and disc diffusion assays against Meticillin-resistant Staphylococcus aureus, Meticillin-resistant S. epidermidis, Escherichia coli, Pseudomonas aeruginosa and Enterococcus faecalis. Antibacterial activity without antibiotic was low, confirmed by high MIC/MBC, and a no inhibition on agar lawns. However, VDAM with antibiotic demonstrated effective antibacterial capacity against all bacteria. Therefore, antibiotic decontamination is a reliable method for sterilisation of AM and the resultant antibiotic reservoir is effective against gram-positive and –negative bacteria.Amniotic membrane (AM) is used to treat a range of ophthalmic indications but must be presented in a non-contaminated state. AM from elective caesarean sections contains natural microbial contamination, requiring removal during processing protocols. The aim of this study was to assess the ability of antibiotic decontamination of AM, during processing by innovative low-temperature vacuum-drying. Bioburden of caesarean section AM was assessed, and found to be present in low levels. Subsequently, the process for producing vacuum-dried AM (VDAM) was assessed for decontamination ability, by artificially loading with  However, the toxic side-effects of antibiotics can be harmful to epithelial cells of the cornea3.Treatment of ocular surface disease, ulcers and infections remains a challenge. Severe bacterial ocular infections are commonly treated with corticosteroids and an intensive course of antibiotics7. Application of AM in the treatment of corneal disease has been reported to reduce infection-causing microorganisms and accelerate healing of infected corneas8, having been shown to be superior to antibiotic treatment alone9.Human amniotic membrane (AM), processed and preserved for therapeutic application is reported to possess anti-inflammatory, anti-scarring, anti-angiogenic and anti-microbial properties, which are valued for their role in wound healing and prevention of post-surgery complications9. Although the mechanism of action of AM is not well explained, it is often attributed to bioactive substances such as growth factors, pro-inflammatory cytokines and anti-inflammatory cytokines, that enhance the function of structural constituents, working as a substrate for local cell migration and adhesion10.AM intervention is an option that mitigates the prolonged use of topical, broad-spectrum antibiotics, preventing steroid-related complications. Studies supporting the use of AM for the treatment of corneal ulcers show significantly enhanced epithelial healing and a reduction in inflammation, corneal scarring, neovascularization, and infection complications11, increased probability of disease transmission, and short shelf-life. Therefore, most clinically used AM is subject to processing and preservation steps prior to use10. Cryopreservation12, heat-drying13 and freeze-drying14 are the most common methods for AM preservation. Although associated with low post-transplantation infection complications15, cryopreservation has been shown to damage the amnion16 which impairs the viability and proliferative capacity of epithelial cells17, decrease levels of many bioactive substances including epidermal growth factors (EGF) and transforming growth factors (TGF), and is constrained by distribution difficulties18. Dried AM followed by radiation sterilisation has been shown to be degraded with reduced growth factor content14. This effect on the stability and function of biological molecules such as growth factors, cytokines and structural proteins, affects the quality of the AM as a biological treatment for eye injury, and justifies the development of an appropriate approach for AM preservation.Freshly collected AM is rarely used for clinical treatment due to post-transplant complications19, AM is often aseptically prepared in order to ensure no contamination is introduced during processing and to limit the spread of any natural bioburden. In vivo during pregnancy, AM is considered sterile, however, potential microbial contamination originates from skin and vaginal flora during delivery of the placenta during birth, with the retrieving staff being an additional source of contamination20. Therefore, any manufacturing process must involve steps to effectively decontaminate such bioburden and allow for long-term preservation of AM.Due to potential degradation by terminal sterilisation techniques21. For dried AM products, vacuum-packing followed by radiation sterilisation is often applied to ensure complete microbial inactivity22.Fresh AM processing generally involves a series of washing steps in physiological solutions to remove raw contamination and blood. Subsequent preservation via heat, freeze-drying, or deep freezing at below −60 °C are applied to suppress water activity to maintain tissue stability, and prevent microbial growth. However, to address bioburden contamination, antibiotics are typically added to the collection medium or storage medium for frozen AMThe sterility and integrity of final manufactured AM is necessary for safe and direct application. However, maintaining AM sterility, quality and functional properties during processing or storage is a challenge. Manufacturing AM in the presence of selected antibiotic/antifungal agents can have several advantageous features, including avoiding deleterious effects on the matrix and biomolecules caused by terminal sterilisation, providing sterility, and inadvertently working as a potential antibiotic reservoir that offers a prophylactic treatment following transplantation.18. This is done by artificially loading AM with high levels of Staphylococcus epidermidis, a skin floral species likely to contaminate amnion23, and investigating the decontamination efficacy of the washing, antibiotic treatment and vacuum-drying steps by counting the surviving colony forming units (CFU) using a spread plate method. The implications of biological and physical antimicrobial activity of fresh AM and the inadvertent antibiotic reservoir effect of vacuum-dried AM (VDAM) against strains of gram-positive and gram-negative bacteria are then studied using minimum inhibitory and biocidal concentrations (MIC/MBC) and disc-diffusion assays.The aim of this study was to validate the microbial decontamination potential of a novel vacuum-drying, aseptic manufacturing technique, and to then investigate further the lasting anti-microbial effects of antibiotics added during processing over long-term storage. We identify the level of microbiological contamination, or bioburden, of elective caesarean section (ECS) AM, and then go on to assess the robustness of decontamination during our optimised, innovative protocol for preserving AM, using vacuum-drying and a broad spectrum antibiotic/antifungal cocktailNeisseria cinerea or Micrococcus spp. Enhanced anaerobic incubation for seven days showed increased growth of Cutibacterium acnes bacteria in samples that were negative at aerobic conditions.Results demonstrating the normal (unloaded) floral contamination of fresh ECS AM can be seen in Table 6 CFU/mL), at different stages of processing, to effectively test the decontamination efficacy of the VDAM process. Figure Due to low and inconsistent levels of natural bioburden on ECS AM, we artificially loaded 5 donated amnion with high bioburden 1006 CFU/mL10 and linear scales). AM samples loaded with S. epidermidis showed significant successive reduction from the original load of 106 CFU/mL as processing progressed. Washing three times in NaCl reduced the bacterial load by almost one log cycle  and the removal of the spongy layer contributed to further lowering the remaining microbial load by 60.53%. Incubating the AM in raffinose solution containing a broad spectrum antibiotic cocktail , P. aeruginosa (ZOI: 2.48 mm) and E. faecalis plates (ZOI: 1.96 mm). All samples of VDAM processed without antibiotic (R) VDAM, processed without raffinose or antibiotic (F), and the PVDF sample discs were not bactericidal to the five strains and did not produce ZOI. However, once the discs were removed there was reduced bacterial growth found below the sample 20. Of those that demonstrated contamination, only Micrococcus spp, Propionibacterium acnes, S. schleiferi and N. cinerea were identified, most likely from skin contact of the placenta during delivery. Previous reports have identified up to 12 species in their surveys including the ones named in our study26. Staphylococcus spp have been identified as the most prevalent contamination of AM26. This study indicates that the risk of major contamination from AM obtained through ECS is low, reducing the risk of contaminated AM being transplanted to the eye. Additional results from PBS samples collected immediately from raw AM before washing (incubated overnight at 35 °C on BA) contained no bioburden (n = 15 from 5 donors), demonstrating that fresh AM could be bacteriostatic before processing.The natural floral contamination of fresh AM obtained by ECS in the Queens’s Medical Centre, Nottingham University Hospitals NHS trust, was shown to be considerably lower than literature reported from normal vaginal deliveriesS. epidermidis were loaded during different stages of processing. S. epidermidis is an ocular pathogen and was selected as a model of staphylococcus genera which is one of the most prevalent causes of bacterial keratitis27. It is considered to be one of the skin floral species most likely to contaminate AM, and has also been shown to adhere well to fresh AM23.VDAM was developed to be stored dry at room temperature in a stable state until use, avoiding the cold chain storage and distribution challenges associated with fresh and frozen AM. A bioburden-free VDAM final product was confirmed during investigation into our protocol, and long-term sterility test results confirm sterility of end-product samples, even when artificially high levels of et al., (2010) who reported that frequent washing can reduce bioburden in processed heart valves28. Most AM preparation protocols use repeated washing in physiological saline to remove blood and immunogens, as well as remains of amniotic fluid. However, we have found that to retain the integrity of the epithelial surface and matrix biomolecules appropriate washing times have to be optimised18. As an added safety precaution for preserved AM, different antibiotic/antimycotic combinations can be used to cover a wide range of gram-positive, gram-negative bacteria and fungi, including penicillin, streptomycin, neomycin, amphotericin B, gentamycin, imipenem, nystatin, polymyxin and vancomycin30. We used a combination of the latter five agents, a cocktail widely used in the UK for tissue allograft banking31. Reports show that antibiotic solutions used for tissue allograft preservation can be stored for up to one-year storage at −20 and −80 without effect on potency. Dried forms of these antibiotics can withstand storage at 37 °C for up to 12 months32.Our results revealed that three successive washes in 0.9% NaCl reduced bioburden by 95.65%, in agreement with Germain 28. Long antibiotic incubation, such as 12 hours at 37 °C, has been shown to be more efficient, but could potentially compromise the cell viability of tissue, including AM34. The current study showed that a two-hour incubation at 37 °C resulted in full eradication of bacteria loaded just before the antibiotic treatment step and that longer incubation times are not required. The antibiotic cocktail was capable of completely eradicating a high load of bacteria.The microbicide activity of antibiotic treatment was found to be more effective at 37 °C than at 4 °C or 22 °CS. epidermidis36. AM can absorb and release antibiotics after rehydration on a wet surface such as agar medium or the corneal surface. Our results showed that the pattern of VDAM release of antibiotic was consistent with the antibiotic control discs, with rapid release at the beginning of application (6 hours) without considerable change in ZOI after 12 and 24 hours of incubation on bacterial lawns. AM has been shown to have slow antibiotic release over seven hours, dependent on the time of AM soaking in antibiotic37, however the release profile may be different for different antibiotics38.AM prepared in antibiotics can serve as a reservoir during transplantation, particularly effective against microbial keratitis frequently caused by different bacteria strains including the conjunctival normal flora 39. AM desiccated to lower or equal to 5% water content prevents microbial growth, thus there is less chance for product contamination. To confirm this, we showed that vacuum-drying alone, without antibiotic treatment, was efficient in removing 94.26% of bacteria, when samples were loaded after the raffinose washing step. Drying in our protocol is primarily applied to AM as a method of preservation and not as a decontamination step, however, so as it was not efficient in reducing all of the bacterial load it would only ever be used as an adjunct to antibiotic treatment for decontamination.Dehydration of AM by air, heat or freeze-drying (lyophilisation) can be applied after washing procedures to decrease the water content of AM, hindering the spread of contamination during storage, and extending the shelf-life. Drying is applied as a final step before packaging, and has been found to maintain AM structural and biological molecules40. No ZOI was observed around washed AM against S. epidermidis38. Tehrani et al. (2013), showed a decrease in antibacterial activity against S. aureus, P. aeruginosa and E. coli of freeze dried AM after 2 hours rehydration, and after 3 times rinsing in PBS41. Our MIC/MBC in vitro study of AM antibacterial activity showed that AM processed without antibiotics has little effect on S. aureus, S. epidermidis, C. diphtheria and Moraxella, indicating that AM has little intrinsic antimicrobial action without the inclusion of antibiotics during the manufacture process. To then investigate the role of the included antibiotics, we then decided to use bacterial strains that may show more resistance to the included antibiotic cocktail in disc diffusion assays. The results of these assays showed antibiotic processed VDAM has a variant ZOI on MRSA (10.74 mm) and MRSE (15.87 mm). Reduced activity was observed on E. coli (8.82 mm), P. aeruginosa (2.48 mm) and E. faecalis (1.96 mm) plates, but was still present and in a consistent manner. AM processed without antibiotic and/or raffinose were ineffective against MRSA, MRSE, E. coli, P. aeruginosa and E. faecalis strains with no ZOI, further indicating that washed AM untreated with antibiotic is not bactericidal.Previous studies have shown that AM antibacterial function after washing or prolonged rehydration was significantly reduced42, and implies that fresh AM may have only a static antimicrobial action by adherence rather than a bactericidal effect, in agreement with Elian et al, 199140. Placing fresh AM on PVDF membrane, to evaluate the biological antibacterial activity by anti-microbial peptide stripping, demonstrated that samples of AM without antibiotics have no such effects against any tested bacteria. These findings disagree with that of N. Kjaergaard et al., 2001, who showed that saline washed AM placed over a nitrocellulose paper was inhibitive to underlying bacterial growth of group B streptococcus, Staphylococcus saprophyticus, P. aeruginosa and S. aureus43.Moreover, visual assessment of confluence of growth underlying the discs showed that bacteria did grow slightly underneath the control discs of plastic, PVDF and untreated AM, but not under VDAM and antibiotic-soaked filter paper. AM antibacterial action by adhesion was higher against gram-positive bacteria compared to gram-negative bacteria. This could be explained by the differences in defence mechanisms between both groups of bacteriaThe current study was limited to investigating whether the antibacterial action of the dried amnion was due to the intrinsic properties of amniotic membrane or due to the antibiotics added during processing. This study could have been further extended by investigating the individual activity of the antibiotics within the cocktail and by considering the effect of long-term storage on each of the individual antibiotics. The study could also be extended by investigating the anti-fungal properties of both amniotic membrane and the antibiotic/antimycotic cocktail added during processing.15. This effect is most likely due to antibiotic use, providing an antibiotic reservoir, substituting for the fresh AM’s lost antibacterial property and effective in prohibiting bacterial growth on wounds44. AM also functions as a physical barrier against infective pathogens45, with elastic, pliable properties allowing it to adhere completely to wounds23, preventing external infection recurrence.AM processed under approved guidelines and as a licensed protocol results in better wound healing results and less post-transplant infectionsThe risk of major bioburden contamination of caesarean section collected AM is low. Nevertheless, the combined AM processing procedure used in this paper is a robust method to remove any bioburden, sub-clinical infection, and manufacturing-related contamination. Non-antibiotic processed AM may be bacteriostatic but has no bactericidal effect. The specific VDAM processing steps of washing in NaCl, spongy layer removal, raffinose incubation and drying, all contribute to significantly reduce the bacterial load, with drying and antibiotic treatment being considered a sterilisation step. The antibiotic reservoir of VDAM can eliminate natural and acquired contamination of gram-negative and gram-positive bacteria, such that VDAM is a reliably sterile product suitable for clinical application. Combined with vacuum packing, VDAM can be confidently and stably stored long-term without the risk of infection-related degradation.46.This study was carried out with the approval of the Nottingham Research Ethics Committee and the study (OY110101) complied with the tenets of the Declaration of Helsinki. Human placental samples were collected from consenting donors undergoing elective caesarean sections. Patients not complying with the inclusion criteria for tissue donation or having a history of antenatal problems were excluded. Written informed consent from the donor was obtained for sample use and the placenta membranes were prepared according to previously published methodologyAM samples (separated from the chorion membrane) from 10 different donors were studied for the natural bacterial flora contamination. Unwashed AM samples were collected in 5 mL PBS  before sonication for 5 minutes. 200 µL of each sample was plated onto blood agar plates  and incubated aerobically for 48 hours and anaerobically for 7 days at 37 °C, before colony forming units (CFU)/mL were counted. 100 µL volumes of sample were inoculated into Anaerobic Basal Broth (Oxoid), for 48 hours and 7 days at 37 °C in aerobic conditions to enhance bacterial proliferation. Bacterial growth was identified by turbidity and API® Staph Rapid 32 A microbial identification kit  was used to identify microorganisms.AM was separated from the chorion membrane by finger blunt dissection and triple washed in 250 mL 0.9% sodium chloride (NaCl) solution  for 15 minutes with rocking agitation to wash out blood. The spongy layer was removed by scalpel blade size 22 attached to a custom-made tool. The AM were soaked in 100 mM raffinose pentahydrate  solution in DPBS containing 5 mL of a broad-spectrum antibiotic/antifungal combination   and incubated overnight at 37 °C. The next day absorbance at 490 nm was measured using a Jenway 6705 UV/Vis Spectrophotometer  and the bacterial suspension adjusted to 108 CFU/mL.A pure culture of a clinical isolate of S. epidermidis was performed individually at three different stages of the VDAM process to assess the efficiency of the decontamination process at different steps . The average of viable colonies was then counted and expressed as CFU/mL.Loaded and fresh AM samples in 1 mL PBS were twice diluted to give 106 CFU/mL of gram-positive; Staphylococcus aureus (S. aureus), S. epidermidis, Corynebacterium diphtheriae, and gram-negative Moraxella lacunata bacterial strains. 50 µL/well of lysogeny broth (LB) medium (Sigma-Aldrich) was added to appropriate wells of a 96-well plate, followed by 50 µL of AM extract at concentrations of 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.20, and 0.10 µg/mL loaded in triplicate across the plate. Culture medium only and medium with 100 mg/L gentamicin were used as negative and positive controls respectively. The bacterial strains were grown up in LB medium until optical density = 0.1 at 600 nm. 50 µL/well of the bacterial cell suspension added to each well of the plate. Plates were incubated overnight at 200 rpm on a rocker at 37 °C. Results were assessed visually with the minimum inhibitory concentration (MIC) equivalent to the last clear well, where bacterial growth is completely inhibited, and the minimum bacterial concentration (MBC) equivalent to the lowest concentration of test article required to kill 99.9% of the initial bacterial inoculum.Dry AM discs from 3 donors were prepared with and without antibiotics. Proteins were then extracted from the tissue by grinding with pestle and mortar in a liquid nitrogen bath. AM extracts were then tested against 10S. aureus (MRSA), multidrug resistant S. epidermidis (MRSE), Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa bacteria were prepared from fresh pure cultures in TSB. The viable bacterial count of each was adjusted to 108 CFU/mL using 0.5 McFarland standard. Each of the five bacterial cultures were seeded onto three Mueller-Hinton agar (MHA) (Oxoid) plates in 90 mm Petri dishes using a sterile cotton swab and subsequently the AM discs were aseptically placed on the even lawns of the bacterial strains. An antibiotic-soaked disc of Whatman no.3 filter paper (AB) was performed as a positive control and a disc of transparent laminating plastic (P) was used as a negative control. Three additional discs were also assessed, using hydrophobic highly stripping polyvinylidene fluoride (PVDF) transfer membrane discs, pore size 0.45 µm , one of which was used alone as a control (PV). The PVDF was then used to separate the MHA surface and VDAM-R (R/PV) and VDAM-F (F/PV) to assess biological antibacterial activity, rather than physical blocking. MHA plates were incubated at 35 °C for 18–24 hours and zone of inhibition (ZOI) were measured in mm using a digital calliper . To confirm whether there was any bacterial growth beneath the samples, sterile disposable loops were used to touch areas under the discs of the same bacterial plates and streak on another Tryptone Soya Agar  plate before overnight incubation at 35 °C.The following AM samples were prepared for disc diffusion assays; VDAM processed with raffinose and antibiotic (VDAM), VDAM processed in raffinose without antibiotic (R) and VDAM without raffinose or antibiotic (F). All samples were cut into discs of 10 mm diameter and kept in vials at 4 °C for a maximum of 4 days before the experiment was performed. Cultures of multidrug resistant To specifically investigate time line of antibiotic release from VDAM samples, ZOI were measured at 0, 6, 12 and 24 hours.Triplicate samples of 25 mm diameter  and 10 mm diameter  VDAM discs were tested for sterility. Discs were either aseptically immersed in falcon tubes containing 40 mL TSB for aerobic bacteria, facultative bacteria and fungus, or placed in thioglycolate broth medium U.S.P.  for anaerobic and facultative bacteria. TSB tubes were incubated at 35 °C with shaking (80 rpm), for 21 days along with negative and positive control tubes. Thioglycolate medium tubes were incubated for 21 days at 35 °C without shaking. Turbidity was used as an indicator of non-sterility and was checked daily. Streaking of 10 µL TSB from each tube was performed weekly on TSA plates, which were subsequently incubated for 5 days at 37 °C and observed for bacterial growth.Statistical analysis was performed using GraphPad Prism version 7.01.


In the last 20 years, efforts were made to optimize antibiotic use in hospitals across the world as a means of addressing the increasing threat of antibiotic resistance. Despite robust evidence supporting optimal practice, antibiotic decision-making remains sub-optimal in many settings, including in hospitals. Globally, resources remain a limiting factor in the implementation of antibiotic stewardship programs. In addition, antibiotic decision-making is a social process dependent on cultural and contextual factors. Cultural boundaries in healthcare and across specialties still limit the involvement of allied healthcare professionals in stewardship interventions. There is variation in the social norms and antibiotic-prescribing behaviors between specialties in hospitals. The cultural differences between specialties and healthcare professionals (1) shape the shared knowledge within and across specialties in the patient pathway, and (2) result in variation in care, thus impacting patient outcomes. Bespoke stewardship interventions that account for contextual variation in practice are necessary. The first time the term “stewardship” was used in relation to antibiotic use was in an article in New Horizons in 1996 . These gThe term stewardship, in the Oxford English Dictionary, is defined as “the job of supervising or taking care of something, such as an organization or a property”. It can also be used in the context of taking care of something that is of value, and it is probably in this context that the term first became associated with antibiotic use: to preserve the valuable efficacy of antibiotic agents. Needless to say, prior to the coinage of this term, initiatives targeting optimization of antibiotic use and the preservation of their efficacy to combat AMR were already in place in healthcare institutions ,2,8,9. IAt its core, the objective of ASP is about preserving the efficacy of antibiotics for use in the future, i.e., the expectation on individual healthcare professionals is to adapt their present behavior and clinical decision-making for a future, intangible reward. This is against the school of teaching in medicine. The modern version of the Hippocratic oath states that doctors should “neither over-treat nor resort to therapeutic nihilism” ,13. In tMost of the research and evidence for stewardship programs in hospitals continues to be from high-income countries ,5,16. ThMore recently, a behavioral change framework called the behavior change wheel ,28 was uToday, in hospitals, the patient care pathway includes a multitude of medical and surgical specialties, pharmacists, nurses, and healthcare managers, who are all working to common goals, but, in the process, may prioritize different policies, agendas, and interim targets. Different specialties have their own language, behaviors, social norms, and values . In an eMost of the published ASP studies report an intervention initiated by members of the traditional ASP teams . ResearcThe term culture is defined and interpreted in many ways ,39,40. SIn the last 20 years, antibiotic stewardship evolved continuously, and is now a global drive, with organizations aiming to implement interventions to rationalize antibiotic use in secondary care. To influence behaviors of prescribers, frameworks from social science research are being used. However, these frameworks often do not include a study of context and culture. To bring about sustainable change in prescribing behaviors, and to optimize antibiotic use, it is first necessary to study how and why healthcare professionals behave the way they do. To do this, it is necessary to undertake research into the cultural determinates of antibiotic use in secondary care.