Human activities, especially dam construction contributed 96% to this change, while 4% was due to climate change. El Niño/Southern Oscillation (ENSO) events are often associated with low precipitation, resulting in low water discharge and sediment load, indicating that changes in ENSO periodicity could affect the inter-annual periodic variations of water discharge and sediment load. As population and economy boom, more dams are being built in the Pearl River basin, and special attention should be paid to the management and mitigation of the effects of dams on sediment load.Global climate change and human activities have important effects on the water discharge and sediment load of the Pearl River. In this study, the water discharge and sediment load were investigated by using hydro-meteorological data from 1954 to 2018. The linear regression, Mann–Kendall abrupt test and double mass curve were employed to detect trends and abrupt change-points in water discharge and sediment load and to quantify the effects of climate change and human activities on water discharge and sediment load. The results revealed that the annual sediment load exhibited a significant decreasing trend at a rate of − 2.24 × 10 Therefore, understanding variations of WD and SL from the rivers to estuaries and oceans has been set as one of the goals of the International Geosphere–Biosphere Programme and its core project, Land Ocean Interaction in the Coastal Zone4. It has been concluded that climate change and human activities are the most important factors influencing riverine WD and SL6. Scientific, observations have indicated that the global surface temperature has increased by approximately 0.8 °C with a significant upward trend over the past 30 years and a rate greater than 0.2 °C per decade7. Global annual precipitation has also increased significantly at a rate of approximately 0.2 mm/year (P < 0.001)8. This global climate change has influenced global and regional hydrological cycles. It has estimated that global runoff could increase by 4% based on an increase in global temperature of 1 °C9. Additionally, human activities, such as land use changes, freshwater extraction, and dam construction, have intensified over past several decades, often resulting in significant influences on river systems6. Decreased sediment loads have caused erosion in many river deltas, including the Mississippi River in United States10, Mekong River in Vietnam11, Yellow River12 and Yangtze River13 in China. The response of water discharge and sediment load to climate change and human activities have become hot topics, garnering significant attention worldwide17. However, the causes for changes in WD and SL differ from river to river and vary over time. Therefore, it is necessary to expand and update knowledge regarding these influences for specific rivers, particularly large rivers, to aid in global and regional environmental management.Water discharge (WD) and sediment load (SL) into the sea are dominant factors controlling beach processes, delta and estuarine evolution, and coastal zone ecological environments22. Some studies have investigated the spatial and temporal variations of WD and SL in the Pearl River basin (PRB). Some attention has been paid to the impact of the soil conservation, deforestation, and dam construction on SL in the PRB. However, most of these studies have primarily focused on the influence of human activities or climate change impacts. More importantly, the quantification of climatic and anthropogenic effects on WD and SL in the PRB have received little attention. In the PR, similar to many other rivers, natural oscillations in the hydrological cycle and the processes influencing such oscillations must be distinguished before possible anthropogenic impacts can be analyzed accurately. The El Niño/Southern Oscillation (ENSO) is a result of ocean–atmosphere interactions on a macro spatial scale and is treated as the strongest inter-annual signal of climatic changes23. Thus, ENSO can be used as a specific parameter to characterize global climate change. Numerous studies have shown that ENSO-driven changes in temperature and precipitation correlate well with mean annual and seasonal river discharge25. Additionally, as global climate conditions changed, the frequency of global ENSO events increased during the late 1970s26. As a result, ENSO has become one of the most potential source of natural variability in the PRB. However, the effects of ENSO events on WD and SL in the PR have rarely been examined. Hence, this study, aimed to (1) provide updated estimates of WD and SL from the PR into the sea, (2) give an in-depth explanation of the influences of ENSO events on WD and SL, and (3) give a quantitative evaluation of the contributions of climate change and human activities to changes in WD and SL at the basin scale. We believe this study will provide a better understanding of natural and anthropogenic contributions to major river water and sediment transport processes, which will provide scientific guidelines for global river management.The Pearl River (PR) Fig.  is the s27. They also strongly affect local- and regional-scale climates base on teleconnections affecting coupled ocean–atmosphere and land systems24. In the PRB, the average annual precipitation over the period of 1954 to 2018 was 1559 mm, and annual precipitation varied between 1120 and 1981 mm. The warm phases of monthly sea surface temperature anomalies (SSTA) were closely related to low precipitation in the PRB  and two periods with increasing trends (1968–1982 and 1993–2001). These findings match the perturbations in precipitation over the 6 decades studied. These results suggest that an increase or decrease in WD could be attributed to climate change and natural climatic oscillations .Figure 8 m3/year and 2078 × 108 m3/year, and lower SL of 1782 × 104 t/year and 1614 × 104 t/year, respectively. These values are lower than the average decadal precipitation, WD, and SL values in the 1960s and 2000s, respectively. Furthermore, the inter-annual variations in precipitation, WD, and SL at time scales of 2–8 years are consistent with the periodic variations of ENSO22. This linkage indicates that changes in ENSO periodicity can affect inter-annual periodic variations in WD and SL. Similar results were also found in Columbia River30, Mekong River31, Yellow River32 and Yangtze River33.Regarding the annual SL, although large changing trends were detected from 1954 to 2018, inter-annual fluctuations were consistent with those of precipitation and WD . The time series for SL can be divided into three phases  with turning points in 1982 and 1988 . Prior to 1982 (linear phase), the cumulative precipitation and SL are well correlated, and precipitation and SL exhibit a significant relationship. During the periods of 1983–1988 and 1989–2018, SL exhibits a significant increasing and deceasing trends, respectively regardless of precipitation and WD. Figure 2 also reveals that for the same level of precipitation, the SL increase from 1983 to 1988 and gradually decrease during the periods of 1989–1998, 1999–2006, and 2007–2018. The linear regression equations for annual SL suggest a rate of increase of 1.38 × 104 t/year from 1983 to 1988 and a rate of decrease of 2.24 × 104 t/year from 1989 to 2018. These opposing trends, which independent of precipitation and WD, suggest that there are external controlling mechanisms influencing SL in addition to natural climate change.The M–K abruptness test and cumulative anomaly test revealed an abrupt change in SL in 1998 Fig. b3. The tThe human activities affecting WD and SL include land use change and dam construction. Typically, soil and water loss induced by deforestation results in an increased SL. In contrast, afforestation, soil preservation, and dam construction result in reduced SL. When both positive and negative influences on SL occur in the same period of time, the magnitude of the measurable change in SL depends on the relative balance the two types of factors.34. As a result, soil erosion in the PRB has been speeded up. Although dam construction on the PR began in the 1960s, the decreasing effect of dam construction on SL did not overcome the increasing effect of deforestation in this period. In the second phase from 1983 to 1988, there was a significant increase in SL. The regression relationships indicate that the SL between 1983 and 1988 was approximately 30% higher than that between 1954 and 1982. This trend is mainly caused by the acceleration of soil erosion. At the end of the 1970s, China implemented in a program of agricultural and economic reform. Land was distributed to peasants and crop planting increased significantly, which further accelerated the deforestation. Xia (1999) indicated that the area of soil erosion in the PRB was almost three times higher in the mid-1980s than in the 1950s35. In contrast, dam construction in the PRB slowed down in the 1980s20. These observations implies that deforestation governed the variation in riverine sediment in this period, which outweigh the influence of dam construction, resulting in an increase in SL. In the third phase from 1989 to 2018, the SL decreased dramatically. The SL in the 2000s and 2010s were only less than half and one third of the 1950s, respectively. This variation, on the one hand, is caused by the efforts to rehabilitate rocky desertified areas, on the other hand, it can be attributed more to an increase in large dam construction. Since the Soil Preservation Law of the Republic of China was enacted in 1991, great efforts have been carried out to prevent soil erosion in Pearl River. According to the remote sensing images of the National Land Use Survey, the area of soil erosion in the PRB in 1995 and 2004 were 62.70 × 103 km2 and 62.73 × 103 km2, respectively18. That is, the area of soil erosion in the PRB was almost unchanged from 1995 to 2004. However, the mean SL in 1995–2004 (54.4 Mt/year) was 30.7% lower than that in 1986–2004 (78.56 Mt/year). Dai et al. (2008) suggested that the area of soil erosion in the PRB was larger in 2000–2005 than in the 1950s and 1960s18. However, the SL in 2000–2005 was only half of the 1950s and 1960s. Moreover, the Bulletin of Water and Soil Conservation issued by the Pearl River Water Resource Committee indicated that the total amount of soil erosion in the PRB decreased at a rate of 47.4 Mt/year from 2002 to 2014, which was only a quarter of the rate of deceleration in SL (184.7 Mt/year). These observations implies that although efforts at soil conservation were responsible for the decrease in SL, dam construction was the main factor causing the reduction of SL. Similarly, it has been reported that soil preservation is the second most important factor for decreasing SL in the Yangtze River33 and Yellow River36 following dam construction. In the PRB, over 9000 dams and reservoirs have been constructed since the 1950s. Figure 8 m3, all of which are scattered across the PRB. From the 1960s to the 1990s, the total storage capacity of the PRB increased slowly, followed by a boom in dam reservoir constructions after the 1990s , the SL exhibits no clear changes. This is mainly caused by the balance between deforestation and dam construction. After the end of two major wars , China entered a period of peace, which resulted in rapid increases in population and deforestationReservoirs typically impound water during the latter half of the wet season (decreasing trend in discharge from August to November) and release water during the driest months (increasing trend in discharge in January and February) to satisfy the demands of agricultural irrigation in the PRB. Based on the seasonal regulation of reservoir water storage in the PRB, the ratios of flood discharge to annual discharge have exhibited a significant decreasing trend over the past 65 years . These extreme monthly values occur approximately month earlier than the corresponding values for the Yangtze River32. This is likely because the PRB is closer to the ocean (South China Sea) compared to the Yangtze River basin. Therefore, in summer, the southwesterly monsoon winds should transport vapor to the PRB earlier than to the Yangtze River basin. Monthly precipitation and WD exhibit no significant changes between the pre-abrupt and post-abrupt periods in the PRB. However, the SL exhibits a significant difference. The SL in the post-abrupt period is lower than in the pre-abrupt period, with the largest difference occurring in summer . This change is a result of dam construction. Furthermore, the SL in winter in the post-abrupt period is greater than that in the pre-abrupt period. This result is largely caused by the seasonal regulation of reservoir water storage.Figure mer Fig. b3. This To quantify the contribution of climate change and human activities on WD and SL variations, a benchmark period must first be defined to represent that this period is mainly dominated by natural processes and the impact of human activities on WD and SL can be negligible. Then, the liner regression method is used to establish the WD and SL for the following periods based on the precipitation data. Finally, the calculated WD and SL can be considered as the contribution of climate change. The difference between measured and the calculated values is considered to be influenced by human activities. As analyzed above, the WD and SL in the PR exhibit no obvious changes prior to 1982. The impact of human activities including dam construction and deforestation during this period was not significant. Therefore, the period from 1954 to 1979 can be used as a benchmark period to quantify the influences of climate change on WD and SL variation. The linear regression equations between the precipitation data and both WD (1) and SL (2), as well as their significance levels, are presented below.8 m3/year) and SL (7955.01 × 104 t/year) were close to the observed data (2763.63108 m3/year in WD and 7783.88 × 104 t/year in SL), with errors of less than 1% and 2.2%, respectively, which also supported the simulated values obtained for the 1980s to 2010s. As shown in Table Figure 8 m3/year relative to the baseline. Climate change and human activities contributed + 109% and − 9% of this increase, respectively. The most significant impacts of human activity on WD occurred in the 1980s, where the contribution of human activities to WD increase was 56%. Deforestation in the watershed will lead to soil erosion and water loss, which will increase water discharge. Generally, in the PRB, climate change contributes much more to hydrological factors, regardless of whether its effects are negative or positive. For example, during 2010s, climate change and human activities contributed 78% and 22% to the reduction in WD, respectively. Regarding SL, the greatest increase occurred in the 1980s, where the value increased by 979.64 × 104 t/year relative to the baseline. The contribution of human activities to this increase was 166%. SL decreased from the 1990s to 2010s. The SL decrease caused by human activities was 2107.17 × 104 t/year in the 1990s, 4121.53 × 104 t/year in the 2000s, and 5638.39 × 104 t/year in the 2010s. The contribution of human activities varies from 96 to 336%, with the most obvious increase occurring in the 1990s. During 2010s, the contribution of climate change and human activities to decrease in SL were 4% and 96%, respectively. These results suggest that the impact of human activities on SL exceeds that of climate change, meaning human activities play a dominant role in terms of sediment change.Table 38. In particular, these large-scale ocean–atmosphere processes will interact and adjust to varying degrees, making the precipitation processes in the PRB more highly complex. Therefore, more attention needs to be paid to the correlation between these processes and their impact on precipitation and WD in future studies.The spatiotemporal variation of precipitation will have an important impact on the spatiotemporal distribution of water resources, leading to severe floods or droughts. In addition to the strong ENSO event, which has significant impacts on regional precipitation in the PRB, other global climate changes and large-scale ocean–atmosphere processes, such as Pacific Decadal oscillation (PDO), Indian Ocean dipole (IOD), and North Atlantic oscillation (NAO), also influence the hydrological cycle of the PR9 m3. However, it was constructed across a tributary of the East River (Xinfeng River), where the catchment area is just 5.7 × 103 km2 and the SL before its operation was only 0.8 Mt/year. Therefore, the impact of the dam on SL in the PR is not significant. Since the beginning of the 1990s, dam construction in the PRB has accelerated significantly. As shown in Fig. Among the human activities in the watershed, dam construction is the most direct way to manipulate the SL. However, the impact of the dam on SL depends not only on its storage capacity, but also on its location on the river, which determines the catchment area, water discharge, and whether there are other dams. In the PRB, although many dams were constructed in the 1950s–1980s, their impact on the SL is not significant because most of them have small storage capacity and are located on tributaries. A few dams with large storage capacity also have relatively limited impact on SL due to the small catchment area. For example, the XFD dam Fig. a, built 39, but also for understanding the evolution of deltas and estuaries, as well as coastal environments34. Dramatic decreases in sediment discharge accelerate delta and shoreline recession, which has been observed in many deltas, such as the Nile River delta40, Ebro River delta41, Red River delta42, Mekong River delta31, Yellow River delta43, and Yangtze River delta44. Therefore, it is reasonable to conclude that anthropogenic impacts on WD and SL have changed the evolutional pattern of the Pearl River delta and its coast. Recently, a deceleration of the delta growth rate has been reported in the Pearl River delta, although the general trend is still prograding45. Most river outlets began eroding in the 1990s to 2000s, rather than silting up as they had previously14. Although there are other factors contributing to these changes, such as in-channel sediment mining and unreasonable coastal construction, the decline of SL is a dominant factor. Furthermore, given the large number of reservoirs in the PRB and their enormous storage capacity, the SL in the main river is expected to remain low on a century timescale18. Therefore, in future scientific research and management projects, increased attention must be paid to the long-term effects of a reduction in sediment flux on environmental changes. Furthermore, the remarkable variation in SL in the PR combined with nearly unchanged levels of WD  has the lowest water discharge and sediment load of the three tributaries. The ‘Pearl River’ water and sediment data in this study are defined as the sum of these three tributaries. Influenced by the subtropical monsoon climate, water discharge levels from the Pearl River fluctuate seasonally and significantly. Water discharge and sediment load during the flood season (April–September) account for ~ 78% and ~ 98% of the total annual amount, respectively.The PRB covers the area ranging from 21.31° to 26.49° N and 102.14° to 115.53° E with a drainage area of 0.45 × 1013 and cumulative anomaly method38 were applied to detect any abrupt changes in annual precipitation, water discharge and sediment load. The double mass curve method23 was used to estimate the relative effects of climate change and human activities on water discharge and sediment load in the basin.The linear regression method was used to analyze the trends precipitation, water discharge and sediment load from 1954 to 2018. The Mann–Kendall test method


Study of the c-Met-HGF axis in non-small cell lung cancer (NSCLC) has focused on the roles of c-MET signaling in neoplastic epithelial cells and the secretion of its ligand hepatocyte growth factor (HGF) by tumor stromal cells. However, there is increasing evidence that some leukocyte sub-sets also express c-MET raising the possibility of an immunomodulatory role for this axis. Consequently, the role of the c-MET- HGF axis in immunoncology is an active area of ongoing research. This review summarizes current knowledge of c-MET expression in NSCLC, the prognostic significance of these findings and the mechanisms by which the c-MET-HGF axis might act in NSCLC, focusing on the emerging evidence for an immunoregulatory role. In the steady state, the receptor is primarily found on epithelial cells where it regulates cell motility and proliferation . The ligIn-vitro studies by Frampton et al., support the conclusion that Exon-14 splice mutated tumors will respond to anti c-MET therapies. Case reports from three patients also provides limited clinical data to support this conclusion  by tumor cells is another pathway by which the c-MET-HGF axis may contribute to an immunosuppressive tumor microenvironment . IDO exeol group . FurtherThe evidence above, indicates that role of aberrant c-MET activity on expression of immunoregulatory molecules by tumor cells is worthy of further investigation in both MET dependent and non-MET dependent NSCLC as is the potential combination of c-MET targeting therapies with immune check point inhibitors.The expression of c-MET is not limited to epithelial cells. The HGF-c-MET axis is involved in the binding, propagation, and survival of hematopoietic progenitor cells  and therNeutrophils are best understood for their role within the innate immune system, serving as short lived, phagocytic cells acting against pathogens such as bacteria . There iin-vitro experiments showed c-MET knock out neutrophils have reduced nitric oxide-mediated cytotoxicity against neoplastic cells, whereas c-MET positive, HGF-stimulated neutrophils demonstrated increased nitric oxide (NO) release and enhanced cytotoxicity  . Althougigration .The studies by He et al., Finisguerra et al., and Glodde et al., suggest c-MET inhibition could become an adjunct therapy to established immunotherapies because they demonstrate a role for c-MET in the recruitment of TANs, show that tumor derived GM-CSF can stimulate increased HGF-production by neutrophils and that HGF can promote increased expression of PD-L1 and PD-L2 on neutrophils. Further work is needed to see if this could be applicable to the treatment of NSCLC. Currently, few studies have investigated expression of c-MET on TANs from human NSCLC. In four NSCLC patients, where neutrophils were isolated from tumor samples and from non-cancerous lung tissue, gene expression of MET in TANs was greater than in neutrophils from healthy tissues . HoweverLymphocytes play a vital role in influencing immune responses to tumors. Whether lymphocytes mount an immune response or a promote immune tolerance toward tumor cells is influenced by a number of factors including, cytokines, tumor cell immunoregulatory molecules, and secreted proteins, as reviewed by Chraa et al. . In addiin vivo and in vitro /generating tolerogenic dendritic cells , 55. c-Min vitro . c-MET pesponses . c-MET iesponses . c-MET pesponses . The prec-MET positive B-lymphocytes in solid tumors have not, to the authors knowledge been reported. However, the HGF/c-Met axis has been shown to play a role in survival of mature murine B lymphocytes via the CD74 pathway , and is Lymphocyte responses can be influenced by other types of leukocytes such as dendritic cells. As there is some evidence of c-MET expressing dendritic cells targeting these cells may also provide another avenue via which c-MET inhibitors would be used to improve immunotherapy outcomes in cancer patients. Dendritic cells (DCs) are the primary antigen presenting cell population responsible for initiating T-cell activation and development of adaptive immune-responses. c-Met expression in dendritic cells has been best studied in inflammatory disease wherein it drives a tolerogenic rather than immunogenic phenotype. In a model of allergic asthma messenger RNA (mRNA) for c-MET was identified in murine DC cells and HGF suppressed the capability of DC to present antigens . HGF hasMonocytes are circulating precursors to macrophages and dendritic cells, which can have immunomodulatory effects on local T-cell responses . c-Met h+, HLA-DR−, CD14+CD15− cells  are a group of leukocytes which act to suppress T-cell effector functions and can be broadly divided into two populations myeloid (M-MDSCs) and polymorphonuclear (PMN-MDSCs) . M-MDSCs5− cells . PMN-MDStrophils . Use of −, CD11b+ CD33+) can constitutively express low levels of c-Met (In human blood, CD14 negative leukocytes including MDSCs (CD14of c-Met . Culturiof c-Met . In addiof c-Met . This suof c-Met .Overall, there is currently little evidence that c-MET expressing leukocyte subsets exist in NSCLC tumors. However, evidence that the c-MET/HGF axis can influence leukocyte activity in the studies discussed above indicates that c-MET inhibitors may potentially be of benefit via their effects on immune cells. This raises the possibility that c-MET inhibitors could be used in patients with non-MET dependent tumors as well as those with tumor cells with aberrant activity.The potential for c-MET to influence both immunoregulatory molecules in MET dependent cancers and leukocytes in either MET dependent or MET independent cancer raises the possibility that treatments targeting the c-MET axis could impact on the outcomes of anti-cancer immunotherapies. Current evidence suggest that immune check point inhibitors are less effective in tumors with driver mutations including c-MET . The conin-vitro and in-vivo results suggest the c-MET inhibitor crizotinib increases immunogenic cell death and thus improves treatment outcomes. Additionally, these effects are related to T-lymphocyte infiltration and depletion of T-cells or blockade of IFN-γ abolishes these effects. As crizotinib effects appeared to be off target this again suggests a potential use of c-MET inhibitors in MET dependent and non-dependent tumors.The c-MET inhibitor crizotinib has been studied as a means of improving treatment outcomes, via an off target effect. Responses to some chemotherapy agents is partly due to causing “immunogenic cell death” which allows immune cells to be attracted to dying cancer cells and progression free and overall survival have been linked to the expression of receptors associated with immunogenic cell death. Crizotinib has been used to induce immunogenic cell death in a human NSCLC cell line . When ceAlthough, the role of the HGF/c-MET axis as a regulator of immune function is incompletely understood it is emerging as a powerful influence in many aspects of immunity. The studies described above have illustrated its effects on first line responders such as neutrophils, in directing the migration and stimulatory capacity of dendritic cells and monocytes through to directly influencing the function of effector T-cells. These effects occur in concert with, and must be considered alongside, direct effects on tumor cells and stromal cells in the tumor microenvironment. As many novel anti-cancer therapies target immunoregulatory molecules or the c-MET receptor itself understanding the relationship between c-MET and immunological responses to tumors is critical in ensuring these drugs are used to their full potential benefit.HT performed the literature searches and wrote the first draft of the article. HT, RO'C, KD, and AA discussed and contributed to manuscript content. All authors reviewed the manuscript before submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A best evidence topic has been constructed using a described protocol. The three-part question addressed was: In patients who underwent appendicectomy for uncomplicated appendicitis is the use of postoperative antibiotics associated with lower rates of surgical site infections?The search has been devised and 6 studies were deemed to be suitable to answer the question. The outcome assessed was the efficiency of postoperative antibiotic therapy in decreasing the rate of surgical site infections in uncomplicated appendicitis. Authors recommend against the use of postoperative antibiotics based on the supported evidence. Hence, its usage was not associated with lower rates of surgical site infections. On the contrary, it might increase the cost, postoperative morbidity and length of stay. A BET p2After performing an appendicectomy for uncomplicated or simple appendicitis for one of your patients. You wonder whether the addition of further postoperative antibiotic therapy would decrease the chances of developing superficial or deep surgical site infections?3In [patients who underwent appendicectomy for uncomplicated appendicitis] is [the use of postoperative antibiotic] associated with lower rates of [superficial or deep surgical site infections]?4The search was conducted as following:Embase 1974 to 2020 and MEDLINE® 1946 to November 2020 using the OVID interface. The results were limited to English articles and human studies:[simple appendicitis OR uncomplicated appendicitis] AND [appendicectomy OR appendectomy] AND [postoperative antibiotic OR post-operative antibiotic] AND [surgical site infection OR SSI OR intra-abdominal collection OR intraperitoneal collection OR pelvic collection].56963 articles were found. Out of these 9 deemed to be suitable and met the criteria of our search. 6954 articles were excluded as they included duplicate, conference abstracts and irrelevant articles based on titles and abstracts. 9 full-text articles were screened and assessed for eligibility. 6 out of 9 articles were chosen as they provided the best evidence and were confined to postoperative antibiotics administration in simple or uncomplicated appendicitis and their role to decrease the postoperative surgical site infections which is the core of our topic. The full articles reviewed carefully to ensure including the best evidence to answer the question.Different papers have defined acute simple/uncomplicated appendicitis differently but all of them have agreed to define it as inflamed nonperforated appendicitis. Surgical site infections (SSIs) defined as both superficial and deep surgical site infections which occur within 30 days postoperatively. Superficial surgical site infections were defined as superficial wound infection which requires antibiotic therapy or laying the wound open along with wound care. Deep surgical site infections defined as the fluid collection inside the peritoneal cavity confirmed by ultrasound or computed tomography that requires drainage or antibiotic therapy.6(Please refer to the table).7Post appendicectomy SSIs is one of the common adverse events affecting patient safety worldwide . PreoperIn 2009, Le et al. [In 2011, Coakley et al.  came up Hussain et al.  in 2012,In 2013, Hughes et al.  devised In 2015 and 2017, both Rafiq et al.  and MoosAll the above-mentioned studies have strongly agreed on the efficiency of preoperative antibiotic usage for decreasing the incidence of SSIs and that postoperative antibiotics were not beneficial in case of uncomplicated appendicitis.The observed limitations are the possibility of selection bias and the variability of the selected antibiotic regimen in some of the studies. However overall, they came up with unified strong recommendations.8Six studies including three randomized controlled trials have strongly recommended against the administration of postoperative antibiotics in case of uncomplicated or simple appendicitis as none of the studies has shown evidence of lower SSIs rate with its use. On the contrary, it might increase the cost, length of stay and postoperative morbidity.Not applicable.None.SA: devised the idea of the study, conducted literature search and wrote the paper.RI: assisted in literature search editing and writing the paper.FA: assisted in literature search and collecting the data.HN: assisted in literature search and writing the paper.AA: assisted in writing and editing the paper.Not Applicable.Name of the registry: not applicableUnique Identifying number or registration IDHyperlink to your specific registration (must be publicly accessible and will be checked)Sabry Abounozha (SA), Rashid Ibrahim (RI), Faisal Mohammed Alshehri (FA), Hossam Nawara (HN), Awad Alawad (AA).None.


Antibiotic resistance is one of the most serious public health risks facing humanity. The overuse of antibiotics in the treatment of infectious disease have been identified as sources of the global threat of antibiotic resistance. This paper examines how farmers perceive and manage risks associated with overuse of antibiotics. Specifically, the paper examines the role of habitus and risk in determining farmers' decisions to adopt national antibiotic reduction targets set by members of the Responsible Use of Medicines in Agriculture Alliance's Targets Task Force. Semi-structured interviews were conducted with 34 sheep and beef farmers in England and Wales. Farmers presented four scripts which illuminated reasons for limited adoption of the targets. The scripts presented the farmers as “good farmers” facing an emerging threat to their ontological security. Scripts suggested that they engaged in preventative measures but deflected responsibility for reducing antibiotic resistance to veterinarians and poorly run farms. This research provides valuable insights for policy makers and highlight the benefits of including social science research to support effective implementation. Antibiotic resistance has been identified as one of the most serious public health risks facing humanity –3. It haCurrently in the United Kingdom (UK), overall veterinary antibiotic use estimates are derived from pharmaceutical sales data . AntibioSheep and beef production are typically extensive industries. There are over 72,000 breeding ewe holdings , and 59,Escherichia coli outbreaks has been risk averse with risk framed as contingent and uncertain  will occur within a stated time frame or as a result of a certain action. Risk assessment is the process of estimating both the probability that an event will occur and the probable magnitude of its adverse effects—economic, health, and safety or ecological over a specified period of time . Zinn 2 suggestsThe way in which expert knowledge characterizes risk suggests that danger can be defined and managed or governed . Risk maemotion-risk assemblage to acknowledge that emotions and risk judgements, rather than being located in the individual, are fluid, shared, and collective.Thus, at a micro level, knowledge production will be understood as situated in specific and contradictory contexts of the everyday. Such embodied knowledge includes pre-rational, aesthetic, emotional, and intuitional aspects of knowledge , 28. SocThe way in which Bourdieu sought to unpack the relationship between individual, agency, and wider social structures as determinants of individual behaviors or practice has been explored in the context of both farmer behaviors and human health –34. CrawThis study builds on the relationship between risk and habitus to gain an understanding of sheep and beef farmers' decisions and actions relating to reducing antibiotic usage, in order to support successful behavior change, policy implementation, and the reduction in the risk of antibiotic resistance.This paper draws on a qualitative study developed as part of a larger longitudinal study which aimed to understand farmers' perceptions and use of antibiotics on sheep and beef farms in England and Wales. Semi-structured interviews were conducted with farmers on 34 farms of which eight were beef-only, four were sheep-only, and 22 were mixed species farms .Farmers who took part in the quantitative study and agreed to be interviewed were contacted by phone to arrange a suitable time for interview. All interviews were conducted on the respondents' farms and fieldwork took place between July 2018 and December 2018. The interviews were conducted by CD, lasted between 30 and 45 min, were digitally recorded and then transcribed verbatim.control a person has or perceives. They make take risks in order to regain control of a situation, or to confirm their level of control over a situation. Level of control is often entangled with trust where social relationships are involved. Peoples risk taking can be driven by their social motives such as feeling of excitement, feelings of self-worth or in response to vunerability. Reflexivity refers to embedded human belief structures rooted in the social world including habitual risk-taking, routinized risk-taking or normalized risk-taking. Finally, a persons social identity can be key to explaining their risk-taking. People may take risks in order to develop their identity, their identity may shaped what risks are deemed acceptable to take or they may take risks to protect their identity. Not all of these domains may be relevant to risk taking regarding farmers antibiotic use or resistance. Instead, we can aim to identify which of these domains are relevant through asking broad questions relating to antibiotic use and resistance. The topics covered in the interviews included general farm practices, antibiotic use, perceptions of current antibiotic use, national reduction targets, perceptions of the risk of antibiotic resistance, antibiotic use on other farms, and perceived responsibility of antibiotic use monitoring. During the discussion of targets for antibiotic use, a show card was used to indicate the national 10% reduction target set by the RUMA Targets Task Force for the sheep and beef sectors .Scripts have been described as moral resources used by farmers to account for and justify their management of disease and as a means of farmers re-constructing their identity . They ar- Antibiotic resistance—the script of an emerging threat- Good farmer habitus—the script of experienced, knowledgeable farmers- Adopting preventative measures—the script of controlling risk and adopting the precautionary principle- Taking responsibility for risk—the script of risk, defense, and otheringWhen asked about their current use of antibiotics respondents reported administering antibiotics for a wide range of problems. In sheep these included mastitis, lameness, watery mouth, abortion, joint ill and navel ill. In beef, antibiotics were mainly used for respiratory disease, lameness, and eye infections.Given the range of infections that were being treated with antibiotics respondents were asked about the potential of developing antibiotic resistance on their farms. When addressing this question they amplified the potential risks associated with antibiotic resistance identifying factors that had the potential to risk not only their reputation as a good, profitable farmer but also to the health and wellbeing of their animals:Well, it could end up catastrophic! I suppose because if you can't treat something that spreads and gets out of hand, I suppose you move onto the antibiotic so you're moving up the ladder all the time onto more expensive drugs, yeah I mean… it'll be bad for both animal and for pain wise, it'd bad for the farmer financially, won't it?” Respondent 26 (Sheep and beef farmer)“Well, they could be very serious if you have a pneumonia outbreak that you can't treat then- what are you going to do? You could lose 30 animals in the blink of an eye. Really so it's devastating potentially if we suddenly found we had no antibiotics that we could use.” Respondent 6 (Sheep and beef farmer)“Obviously, antibiotic resistance would have a massive impact then on the long-term profitability of farm animals. It is something that we have to be very, very careful—through overuse or through incorrect administration, under dosage we could contribute to antibiotic resistance. Weigh your animal before administering so that you are administering the correct dosage.” Respondent 11 (Sheep and beef farmer)“Antibiotic resistance was perceived as a potentially catastrophic risk that would need ongoing risk assessment and management to protect the animals and their livelihoods.Twelve respondents also expressed fears that they may already have observed signs of resistance on their farms and over half felt they were at high risk of developing resistance given they had encountered difficulties in treating infectious diseases in their animals:When we used to have bottles they'd last for… they'd go out of date before we use ‘em, now it’s sort of when you start to use ‘em you use ‘em and 20% of times you use ‘em they just don't have effect now we're starting to see; the antibiotics haven't got the fight power against the disease what they used to have.” Respondent 2 (Sheep and beef farmer)“I bought a calf several years ago now which had resistance to Marbocyl, ‘cause it had scours, resistant to Marbocyl, and caused quite a few problems until we found out what the problem was and obviously got on top of it and it's been fine since, but yeah, I don't want repeats of those situations.” Respondent 17 (Sheep and Beef farmer).“Their scripts suggested that the Specter of antibiotic resistance was challenging their sense of identity as a good farmer undermining their confidence in their ability to identify and treat infections with certainty and challenging their ontological security i.e., their sense of continuity and order in life , 41. TheI did think when I bought the bunch of sheep that introduced the CODD onto the farm, I was thinking we were getting resistance to Oxytetrin or Terramycin, whatever you wanna call the drug, because the sheep's feet… when we were treating lame sheep they weren't responding to the treatment but it wasn't that actually, it was the fact that we were not treating footrot anymore and we were using a drug that didn't really control CODD. Anyway, but it just took me a little while to cotton on to the fact that I was treating a different problem. So we tend to use Zactran now which is very expensive but very effective.” Respondent 6 (Sheep and beef farmer).“And they haven't cleared up, but I don't know if that's resistance or not or just I'm probably assuming it's one type of lameness and it's another type that that antibiotic isn't, yeah. So no, I don't know if we've got any resistance to be honest.” Respondent 21 (Sheep farmer)“Respondents were concerned that rising antibiotic resistance due to inappropriate use in the industry may cause restrictions on what and how antibiotics were used on farms. This posed a threat to the farmer's way of life and the welfare of their animals. Farmers felt that it could get to the stage where only veterinarians could administer drugs to the animals on their farm. This could cause a breakdown in the trust between farmers and veterinarians, or not allow trust to develop to begin with.Even if legislation came in that we had to have a vet to administer antibiotic use that would definitely cause a huge maybe reduction in the use of antibiotics. Economically it's not possible to profitable in keeping sheep and having your vet out to treat or administer antibiotics for every six sheep, so I would have thought then that there would be massive welfare issues, especially within the sheep sector if antibiotic usage was restricted to veterinary surgeons only.” Respondent 11 (Sheep and beef farmer)“But it's going to get very awkward when it's lambing time and you need Pen & Strep or something for a ewe with a difficult lambing and you've got to go all the way down to (town) and get it in a syringe and bring it all the way back again. That's the death of farming. They've got to trust us.” Respondent 15 (Sheep and beef farmer)“Farmers were also concerned that rising antibiotic resistance within the sheep and beef sectors could damage the public perception of agriculture and consequently they could face blame for antibiotic resistance in humans.It's also the public perception of agriculture as a whole; if we're seen as a hotbed for developing resistance to antibiotics how long is it before that transfers to a human population?” Respondent 31 (Sheep and beef farmer)“Overall, respondents presented a story of a major risk or threat to the health of their animals which was changing their relationship with risk, undermining their confidence in their ability to manage the risks encountered and threatening their ontological security—that is their sense of continuity and order in life and in the consistency of the surrounding social and material environment .Habitus is formed in the context of people's social locations and inculcates them into a world view which is based on and reconciled to their position, thus serving to reproduce existing social structures , 33. HabMost respondents reported that they felt that they had the skills to identify infections and administer antibiotics or common conditions that they encountered, such as pneumonia or footrot. They argued that they were experienced stockpersons and capable of assessing symptoms, identifying the disease, and deciding on a course of appropriate action. They reported that their assessments drew on their intuition, situated knowledge and experience to assess risk to the health of the animal:Well it's just years of experience of doing it really, you just know when an animal's ill and it needs antibiotic treatment….It's either severely lame or got an infection from a wound or has got breathing difficulties…So an animal that stands out to us absolutely as having pneumonia we put in the crush, you can see its breathing, you can even listen to it if you wanted to. If you're sure you know what you've got, we would treat that ourselves…Take its temperature, usually if it's pneumonia it'll have a high temperature which will give you an indication of infection there, so yeah you know what it is.” Respondent 1 (Beef farmer)“Well, if you've been around animals for long enough and if you've been brought up on a farm most farmers can identify a sick animal at a first glance. Yeah. You only need to look at their ears and eyes really, and the way they walk.” Respondent 11 (Sheep and beef farmer)“They suggested that most of the time their knowledge and experience was sufficient for them to decide if antibiotics were needed and that their confidence levels in terms of using antibiotics were high:I feel very confident that I know when (to give antibiotics) 90% of the time I'll know when to use them and the 10% of the time I am unsure I have full confidence in my vet.” Respondent 11 (Sheep and beef farmer)“Oh, very confident because I know what a lame sheep looks like.” Respondent 25 (Sheep and beef farmer)“The treatment of common animal illnesses such as lameness and pneumonia were constructed as an ordinary event and part of the everyday activities on farms. The identification and treatment of such common diseases was learned through their upbringing on farms and embedded in the cultural knowledge of farming:Antibiotics, just footrot in sheep, this time of year it's one of my biggest tasks probably doing sheep work; every time you get a bunch in there's always a sheep lame.” Respondent 17 (Sheep and beef farmer)“P2:You're always going to get lameness.“P1:You can identify the lameness so I suppose you'd have to say lameness. You get other things crop up from year to year but lameness will always be… or is always just an underlying one.” Respondent 22 (Sheep farmer(s))Farmers considered they were experts in identifying and treating common diseases. They felt that treatment practices had changed very little over time and that it was very rare they would encounter a disease that was unknown to them. Hence, they seldom called their veterinarian out to visit the farm.There's no new real need for them to come out to sheep… You know, if you have a lame sheep you know what it is and we've had vets in the past tell us what to do so it doesn't really change.” Respondent 10 (Sheep and beef farmer)“Not in general healthcare I wouldn't say, maybe on something new because obviously there's the price of the vet and he's only going to probably say the same thing as what we would do, but if it's something new and we didn't know, yes the vet should be involved to understand it.” Respondent 9 (Sheep and beef farmer)“Farmers believed that their veterinarians trusted their capabilities to identify and treat their animals. For example, one farmer expressed that their experience as a farmer meant that their level of knowledge on their animals' health was on par with less experienced veterinarians.I had a cow last year in the cubicles, she was unwell, she got quite irritable and I rang up the vets and said, ‘Look, I don't know what this is.’ The head vet came out, the senior vet because he said, ‘You know, if you don't know what's going on then there's no point in sending one of the junior vets.”’ Respondent 13 (Sheep and beef farmer)“Respondents painted a picture of themselves as self-assured, competent, and highly experienced farmers who were capable of managing disease risks appropriately. They were able to draw on a range of internal resources to support their decisions. Such resources were part of a collective of skills and expertise acquired as part of their habitus.Shortall et al.  suggest It was evident that respondents feared developing antibiotic resistance believing it would have an extremely detrimental effect on their businesses and their animals. For farmers their farm work represented a central aspect of their life. Any changes to their routine practices needed to be considered carefully:It isn't a case of them feeling that by doing something might risk their business and in farming risking the business is also risking their life, so the consequences are far greater in farming.” Respondent 24 (Sheep and beef farmer)“The prospect of antibiotic resistance was considered risky enough for farmers to start adopting new practices to minimize this risk. They reported using alternative practices in order to protect their animals and reduce the need for antibiotics. They had begun to think about or use alternative strategies for managing disease on their farms. Strategies they reported included:Improving biosecurityWell, the calves—sourcing from clean herds that give good colostrum and good healthy calves and do not mix them with any other animals from any other farms and have them in separate sheds, separate places” Respondent 2 (Sheep and beef farmer)“Managing animal healthFarmers reported managing animal health by using alternatives to antibiotics including vaccines to prevent disease, anti-inflammatory drugs to aid with pain relief and culling to avoid breeding genetically prone animals and stop disease spread:Vaccines as much as you can. That's our theory. I know people say they cost a lot of money but just so much pleasanter if they're not ill, isn't it? No, we've just got into the habit of vaccination.” Respondent 26 (Sheep and beef farmer)“Sheep wise I think you have got to cull the worst ones, possibly the carriers of any disease, footrot and things like that ….I don't know I can't really.Yeah, there's not much else you can do is there?” Respondent 30 (Sheep and beef farmer)“Cause I use anti-inflammatories as well so I try and cut down the use of antibiotics by using anti-inflammatories to help and hit ‘em hard in one go rather than continuous use of antibiotics. I try not to keep injecting with antibiotics.” Respondent 3 (Sheep and beef farmer)“In some cases it appeared that efforts to keep antibiotic use to a minimum had a negative impact on animal welfare. This was particularly the case for the treatment of lameness in sheep. Although prompt treatment of all sheep with footrot with injectable antibiotics is recommended by industry , some faWe only use antibiotics for the ones with maggots in ‘em and the hoof falling off.” Respondent 29 (Sheep farmer)“Footrot is the major bugbear but like I said, we inject if they're really, really bad, if not you just trim and spray it and put ‘em through a footrot bath. Yeah, we're not ruthless with it.” Respondent 30 (Sheep and beef farmer)“Respondents presented a picture of themselves as farmers who were actively engaging in preventative measures, who were signed up to the “precautionary principle” as a way of managing risks associated with infections amongst their animals. Nevertheless, the strategies mentioned did not appear to be part of a coherent plan to manage risk but rather a risk reduction menu from which they could choose a risk reduction option. In adopting these strategies they sought to maintain their sense of ontological security, their self-identity as a good farmer.Targets are a set of directed principles to identify the individual steps necessary to achieve a common goal. Given the level of concern about antibiotic resistance expressed by the respondents targets could potentially provide a benchmark for action, foster accountability and support them to achieve the goal of reducing antibiotic resistance on their farms. A target to reduce antibiotic use by 10% was set for the sheep and beef sectors. However, when respondents were asked if they considered the specific reduction aims within the RUMA targets to be feasible for sheep and/or beef farmers—they identified a range of challenges that they might face in attempting to achieve the targets. Though the RUMA targets are for sector-wide antibiotic use and are aimed at a national level, most respondents reflected on this through their own individual farm use.The notion of having to reduce their antibiotic use by 10% was not an idea that resonated with respondents as they believed that their usage was already lower than the target set. They provided a number of justifications for challenging the idea that they needed to reduce their use and for deflecting responsibility to others.They defended their position as very low users of antibiotics who had already made such changes and argued that they would not be able to successfully reduce them any further.Reduce mine—I don't think I could reduce it much more than I'm doing because I would say I'm probably using a very, very small percentage to most sheep farmers already because we've stopped using it. Yeah, we've already stopped using it in that sense. I mean I'm sure we could reduce it even more by probably making it even cleaner in the lambing sheds. So reducing what we're using I'm sure I can reduce it more going forward but to reduce it by 10% is probably a bit extreme because I'm not actually using that much anyway now because we changed our practice probably five years ago already. But obviously we'll carry on and try and reduce it, reduce it, reduce it but 10% is quite a lot when you're only probably using four bottles a year.” Respondent 34 (Sheep farmer)“Well, 10% of a lot would be achievable but when you don't use a lot I think it becomes more difficult. So we'll have to wait and see on that one. I think it could give us problems in that we're starting at a low point anyway, so as I say, we'll have to wait and see”. Respondent 13 (Sheep and beef farmer)“Antibiotics were reported as being used as a last resort or only in emergency situations so any attempt to further reduce their use further would put their animals' health and welfare at risk:I couldn't really reduce it at all without losing stock- I only really use antibiotic when an animal is ill so it would result in loss of animals.” Respondent 4 (Sheep and beef farmer)“Well, we only use them when they're necessary so you're gonna have problems with disease spreading and having a worse problem.” Respondent 8 (Beef farmer)If you get a major breakdown of, say for instance pneumonia, you've gotta treat and it's out of your control and you've just gotta run with it because the welfare issue is the priority rather than the reduction. It's nice to have a reduction but the overwhelming animal welfare is priority to that.” Respondent 28 (Beef farmer)“The alternative of risking further or ongoing disease amongst their animals was not a risk that they could justify. Nor was a potential risk to their reputation that could result from allowing sick animals to remain untreated. Such action was considered irresponsible in terms of animal health. Their responses appeared to be based on an emotional attachment to their animals and a fear that they could be seen as irresponsible or bad farmers:I think it would be quite bad practice not to use what I use now because it would look like I wasn't caring at all. So somehow there's a line between using it irresponsibly and using it because you need to.” Respondent 34 (Sheep farmer)“It doesn't look very good ‘cause we've got a lot of footpaths and there's always somebody looking over the fence so you've gotta be careful what you do and it's in the best interests of the animal to be healthy and walking around on four legs instead of three.” Respondent 2(Sheep and beef farmer)“I do worry that people looking from the outside say, “Actually, he's got lame sheep there, he's not treating them“”. Respondent 17 (Sheep and Beef farmer)“Respondents reported additional challenges that they felt would prevent them from protecting their livestock. They suggested that they did not necessarily know the risks they were taking when “buying in” animals that were sick or carrying resistant organisms and this made it more difficult for them to optimize or reduce their antibiotic use for newly purchased animals due to a lack of information about their disease and treatment status:Livestock is moving between farms all the time so you don't know what you are buying in when you buy breeding stock, so yes, I mean it is a concern yes.” Respondent 6 (Sheep and beef farmer)“I wouldn't know that, would I, ‘cause you don't get a history of what animals are treated with when they come.” Respondent 19 (Beef farmer)“I guess the problem we have is that we no control on what happens before they come on farm, I think a lot problems, particularly with pneumonia I think are historic.so if they happen to have pneumonia when they're younger they're more susceptible to get it later on in life, aren't they? …There's no point saying 10% less when someone's probably using 20% more than us, that'd make a 30% difference.” Respondent 8 (Beef farmer)“Importantly, trying to interpret and measure progress against the targets, particularly without knowing their baseline usage was considered outside their remit or skill base so they would not be able to meaningfully reduce the risk associated with over use of antibiotics:So as a farmer I don't know what 10 milligram per PCU means.” Respondent 14 (Sheep and beef farmer)“I don't think everyone knows what we are using now to be able to reduce it by 10%. I don't know how you're gonna use the target you've got at the moment ‘cause we haven't really got a baseline target at the moment.” Respondent 5 (Beef farmer)“Yeah, depends on what your usage level currently is, if it's really low then it's harder to get it down by 10%, isn't it? We're always trying to reduce it for sure, because it reduces costs and sick animals don't perform. So, if you can prevent ‘em from being sick in the first place it's a win-win situation. Yeah, I'd have to… we probably can reduce it, whether we can do it by 10%—if we were a big user, you know, there's a lot of farms where it's easily achievable.” Respondent 32 (Sheep and beef farmer)“Having to interpret statistics so they could reduce their antibiotic use by 10% would act as a barrier to change and potentially be at odds of their habitus and their concept of themselves as good farmers who prioritize the well-being of their animals and the viability of their farm. A lack of evidence or information about how the targets were being measured prevented them from being able to decide if they were contributing to achievement of the national target to reduce usage. They reported feeling unable to fully understand the nature of targets and how to turn them into action. In such circumstances, it was difficult to “own” the targets and they sought to locate responsibility for achieving them with others.Given the challenges respondents reported in attempting to reduce antibiotic use they sought to shift responsibility for the achieving the targets and blame for not achieving them on to others. Othering involves defining or defending self-identity by distancing oneself from individuals or other groups who are excluded or regarded as posing risks to self-identity . IndividFarms that overused antibiotics were framed as poorly run and held responsible for increasing the risk of antibiotic resistance:Poor management. Simple as. Not knowing what they're doing properly. Probably not seeking professional advice through their vets. So, using excess, did you say, of antibiotic? Well, if they've got a major problem then perhaps on certain years they've got to, I mean who's to say I might have watery mouth next year. But, generally that seems a bit like poor management, poor husbandry and not consulting the vet enough. I don't know.” Respondent 21 (Sheep farmer)“Too high a stocking densities, mixing of age groups within buildings, unvaccinated animals coming, where they haven't got a clue where they've come from, poor hygiene and general standards so cattle aren't looked after well… full of rats and other things and various things that can spread disease and issues. Blanket antibiotic treatments and stuff like that.” Respondent 1 (Beef farmer)“Well the ones that do blanket treatment for abortion first of all. There are others that routinely use Spectam in every new-born lamb and penicillin when it could be achieved through better nutrition for the ewe. Well, if you have problems with mastitis that could be bred out, that is… you've not to chase yields as much and to not breed from ones who've had mastitis in the past, that could be reduced in the dairy industry. Yes, there are people using too much, definitely. And there are some who under… don't give the prescribed dose, give a half dose and I'm sure that increases resistance, does it?” Respondent 10 (Sheep and beef farmer)“In describing poorly run farms as problematic, respondents were able to distance themselves from this type of risky behavior, even though some of the problems they attributed to others had also been described in their own experiences of the difficulties they faced in achieving the targets. Other farmers were identified as the risky others—who posed a threat to farming. Poorly run farms became unsafe places which threatened animal health and farmers' livelihood.Responsibility for reducing the risk associated with antibiotic resistance was also ascribed to veterinarians. In the UK all antibiotics for veterinary use must be prescribed by a veterinarian. The Royal College of Veterinary Surgeons (RCVS) Code of Professional Conduct states that “The animal or herd must have been seen immediately before prescription or recently enough or often enough for the veterinary surgeon to have personal knowledge of the condition of the animal or current health status of the herd or flock to make a diagnosis and prescribe” . Whilst,The vet is not involved generally because if you get an animal sick when I go round them in the morning I'm not going call the vet before I do it (administer antibiotics). If we've got a really sick animal that the vet has come out to then that becomes the vet's choice but it is very unusual that we'd have a vet out to an animal.” Respondent 34 (Sheep farmer)“But sheep farmers don't keep running down the vets, you know, we can sort out our own lambing, we hardly ever go down for a lambing. The vet comes out for a TB test or maybe a calving but they're always too busy to talk to you.” Respondent 15 (Sheep and beef farmer)“Although they reported having little contact with their veterinarians, when identifying and treating infections respondents suggested that veterinarians should shoulder responsibility for helping farmers to achieve antibiotic reduction targets:No, the vets are the only ones that are gonna tell you that. And hopefully by doing this you're gonna tell me whether I am or not (over using) … the vets gonna monitor it anyhow ‘cause you've gotta buy the antibiotics from the vet, you can't get it any other way.” Respondent 2 (Sheep and beef farmer)“Well, I would've thought the vet would be the best in the know, won't they? They provide us with the antibiotics so they're in a good position, aren't they, to monitor antibiotic use.” Respondent 19 (Beef farmer)“In attributing this responsibility to their veterinarians, respondents appeared to be deflecting responsibility away from themselves alone suggesting that vets should play a significant role in the process. They believed that veterinarians were more knowledgeable about antibiotic use and in control of the situation because antibiotics must be prescribed by them. However, some farmers noted that antibiotics could be too easily accessible from the veterinarian, and some were confused about the conflicting information received from veterinarians.It's too easy to go to the vet and ask for something and they just give it to you and can be used pretty easily.” Respondent 17 (Sheep and beef farmer)“So I know that there are vets that perhaps maybe that give out the antibiotics without good reasoning and without a good understanding, so.” Respondent 1 (Beef farmer)“Sometimes we're slightly conflicting ‘cause we wouldn't have always vaccinated lameness, we'd have trimmed and put them in a footrot bath, unless they were really bad and then we would've, but now we're told we've got to use antibiotics. So it's sort of slightly conflicting information coming out, isn't it?” Respondent 21 (Sheep farmer)“Respondents presented a story of themselves as good farmers who already met the targets set and who would be putting their animals and their reputation at risk if they tried to reduce them further. They portrayed themselves as responsible actors and distanced themselves from accountability for reducing antibiotic use by shifting blame onto poorly run farms and responsibility on to veterinarians. Thus, their responses to risk sought to maintain symbolic boundaries with the farming community particularly in relation to self and others .This is the first study to the author's knowledge to provide insights into the way in which sheep and beef farmers in England and Wales view the feasibility of antibiotic reduction and the risks posed by antibiotic resistance. The present study used a qualitative approach and aimed to build on the relationship between risk and habitus to gain an understanding of sheep and beef farmers' decisions and actions relating to reducing antibiotic usage. Through gaining this understanding, the study aimed to support successful behavior change, policy implementation, and the reduction in the risk of antibiotic resistance. The aim was not to quantify opinions and the results do not suggest that every opinion reported is held by every sheep and beef farmer in England and Wales. However, using this approach we were able to capture the perceptions, beliefs and motivations that underpin behaviors regarding antibiotic use and the potential for its reduction.Lupton  argues tPolicy makers are increasingly acknowledging that the elimination of all risk presents a major challenge. Focusing on systems that more accurately identify and categorize risks and provide programmes for handling and reducing risks are considered more likely to be effective . The insTargets are just one means to achieve progress against a priority, but not all priorities lend themselves to a target. Before deciding on a new national target or whether to have a target at all, policymakers need to consider whether it is the most effective and appropriate means of achieving the desired outcome . Berry eFirstly, is there a widely RECOGNIZED and pressing problem, which requires policy action at a national rather than just local level?Globally and nationally there is a persuasive need to address the unnecessary use of antibiotics to ensure responsible use in humans and animals (both production and companion). The UK sheep and beef sectors are traditionally low users of antibiotics but, nevertheless, have some “hotspots” requiring action including lameness, abortion, and neonatal diseases in sheep and pneumonia in beef cattle. Additionally, the usage figures taken from convenience samples and reported in the latest VMD and RUMA reports suggest that usage in parts of the beef sector may be higher than the dairy sector .antibiotic resistance as an emerging threat that the sheep and beef farmers interviewed in this study were aware of the risks associated with antibiotic resistance stating that it could be catastrophic both for their livelihoods and reputations if they were to develop resistance on their farms. Some feared they may have already observed antibiotic resistance, and most felt at high risk of experiencing it in the future.It was evident through respondents scripts on Secondly, is the problem likely to be amenable to action by those who are accountable for the target?experienced, knowledgeable farmers and of risk, defense, and othering, provided a strong rationale for not cutting back on antibiotic usage. They believed that they were very low users already—only using them in life and death situations—and that that a reduction in use would increase the mortality in their flocks or herds and in turn, potentially risk their reputation as a good farmer. Given that they reported that they were not able to determine their exact use or calculate a 10% reduction their beliefs were based on subjective views.Farmers in their scripts of controlling risk and adopting a precautionary principle suggested that they were signed up to the “precautionary principle” and that they had adopted alternative measures to reduce the risk of infection on their farms.Thus, while it was evident that the problem of antibiotic resistance may be amenable to action by the respondents their strong beliefs about the individual animal being a priority and their concerns about being categorized as a bad farmer may result in action that will limited the potential to reduce antibiotic resistance. Nevertheless, their scripts on However, they did not appear to sign up to the idea that they could be held accountable for the overuse of antibiotics in farming or adopt the target of reducing their antibiotic use by 10%. Their risk rationales were fluid, relational, and contextual . The farExperts' attempts to change risk taking behavior often fail as they do not engage with peoples' identities, the social rooting of risk taking and the social power structure. Respondents suggested that farmers who had more intensive systems, or had poorly run farms with high stocking rates were most likely to be the ones who were creating the risk associated with antibiotic resistance. The act of other-blaming around the responsibility of antibiotic resistance by both livestock farmers and veterinarians has recently been highlighted by Golding et al. , where bThirdly, do the necessary resources to take action already exist or can they be developed?taking responsibility for the risk they explained that when they bought in new animals they were not supplied with their health and vaccination history making it difficult to decide on the action to take if the animal became ill. Availability of medicine records could be improved to make it easier for farmers to inspect the medicine history of potential animal purchases and this should improve as plans develop for the UK centralized medicine hub. A livestock information programme is also being developed through an industry-government partnership in order to improve animal traceability . The patients/participants provided their written informed consent to participate in this study.CD, AR, and JK contributed to conception and design of the study and the themes generation. CD performed data collection. CD and AR performed analysis of transcripts. AR and CD wrote the first draft of the manuscript. JK wrote sections of the manuscript. FL, CH, and LK contributed to the manuscript revision and interpretation of data. All authors read and approved the submitted version.FL is a member on the RUMA Target Task Force as a representative of the sheep industry and LK is an employee of AHDB, which is also a member of RUMA. FL and LK did not participate in design and analysis of the study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


With the growing global threat of antimicrobial resistance, novel strategies are required for combatting resistant pathogens. Combination therapy, in which multiple drugs are used to treat an infection, has proven highly successful in the treatment of cancer and HIV. However, this practice has proven challenging for the treatment of bacterial infections due to difficulties in selecting the correct combinations and dosages. An additional challenge in infection treatment is the polymicrobial nature of many infections, which may respond to antibiotics differently than a monoculture pathogen. With the growing global threat of antimicrobial resistance, novel strategies are required for combatting resistant pathogens. Combination therapy, in which multiple drugs are used to treat an infection, has proven highly successful in the treatment of cancer and HIV. However, this practice has proven challenging for the treatment of bacterial infections due to difficulties in selecting the correct combinations and dosages. An additional challenge in infection treatment is the polymicrobial nature of many infections, which may respond to antibiotics differently than a monoculture pathogen. This study tests whether patterns of antibiotic interactions  in monoculture can be used to predict antibiotic interactions in an obligate cross-feeding coculture. Using our previously described weakest-link hypothesis, we hypothesized antibiotic interactions in coculture based on the interactions we observed in monoculture. We then compared our predictions to observed antibiotic interactions in coculture. We tested the interactions between 10 previously identified antibiotic combinations using checkerboard assays. Although our antibiotic combinations interacted differently than predicted in our monocultures, our monoculture results were generally sufficient to predict coculture patterns based solely on the weakest-link hypothesis. These results suggest that combination therapy for cross-feeding multispecies infections may be successfully designed based on antibiotic interaction patterns for their component species. Staphylococcus-mediated skin infections ; three were predicted to antagonize , and four to interact additively or independently in oculture .E. coli and Salmonella enterica. In this system, an E. coli methionine auxotroph produces acetate from lactose, and an S. enterica evolved mutant consumes the acetate while producing methionine  to identify interactions between drug pairs. We first tested for antibiotic interactions in monocultures of sistance –44. We histance –. We therIn this study, we found that only three antibiotic combinations showed nonadditive interactions; however, our weakest-link hypothesis successfully predicted coculture growth and antibiotic interactions in these cases. Most importantly, we demonstrated that cross-feeding interactions can change antibiotic interactions from either monoculture. While more antibiotic combinations and a greater diversity of cross-feeding partnerships should be explored, these results suggest that the responses of individual community members to combination therapy might be sufficient to predict the antibiotic interactions in the larger microbial community.E. coli , and for streptomycin/ciprofloxacin, spectinomycin/streptomycin, streptomycin/doxycycline, and spectinomycin/doxycycline (where E. coli is the most susceptible). Coculture predictions were somewhat more complicated for the other combinations , where each species is the most susceptible to a different antibiotic. We were particularly interested in nalidixic acid/streptomycin, as these antibiotics synergize in E. coli (most susceptible to streptomycin) and interact independently in S. enterica . Based on the differences in MIC in these species in each antibiotic , we predicted an independent interaction in coculture. Similarly, in the ciprofloxacin/bleomycin combination, the antibiotics verged on antagonizing in E. coli and interacted independently in S. enterica; however, their MICs were similar in both antibiotics. This provided an opportunity to examine interactions in coculture where weakest-link dynamics might play less of a role.According to our predictions, if one species is the most susceptible in both antibiotics, the coculture interaction typically matched that of the most susceptible monoculture. This is the case for nalidixic acid/bleomycin and nalidixic acid/ciprofloxacin (where P = 0.37), we predicted an independent interaction and observed an antagonistic interaction. Interestingly, the isobologram suggested that antibiotics antagonized much more in coculture than we predicted. This suggests that weakest-link dynamics may not always predict coculture outcomes and that some other factor may be determining antibiotic interactions in this case. Quantitatively, our FICI predictions also matched that of our observed data , with one exception. The predicted FICI for the nalidixic acid/spectinomycin combination was significantly higher than predicted (P = 0.037), but this difference still resulted in independent interactions and so is likely not biologically significant. Overall, weakest-link dynamics were generally sufficient to both qualitatively and quantitatively predict antibiotic interactions in cocultures.After generating predicted FICIs based on our monoculture results and weakest-link dynamics, we tested antibiotic interactions in coculture. We then compared our predicted FICIs to those observed experimentally for each antibiotic combination. Qualitatively, our predictions based on the weakest-link hypothesis were accurate—the antibiotic interaction category (antagonism/synergy/additive) identified by predicted FICIs matched the interaction category identified by the observed FICIs  will not always lead to additive drug interactions, however. We saw this in the difference between predicted and observed results for the interaction between spectinomycin and doxycycline in coculture. Most other drug combinations were additive in each monoculture and therefore remained additive in coculture. Further tests of the weakest-link hypothesis for drug combinations with nonadditive interactions are certainly warranted to further verify our results. However, our results provide broad support for the utility of the weakest-link hypothesis as at least a null model when predicting drug interactions in cross-feeding systems.In spite of these challenges, we found that we were able to qualitatively predict the nature of drug interactions in a community context. Our predictions were informed by the weakest-link hypothesis, which posits that the most susceptible member of a cross-feeding system sets the antibiotic sensitivity for all members. We show that weakest-link dynamics should tend to ablate antagonistic/synergistic antibiotic interactions . In lineE. coli was the most susceptible to both streptomycin and spectinomycin, we predicted that the drug interaction in coculture would match the interaction in E. coli monoculture. Instead, the degree of antagonism increased in coculture. These particular antibiotics, which disrupt protein biosynthesis . Additionally, we placed permeable membranes on top of our plates to avoid evaporation and kept our incubator humidified. The first row and column of each plate were antibiotic-free wells for the vertically and horizontally distributed antibiotics, respectively. The MICs for each antibiotic were determined in the absence of the other antibiotic. Mid-log-phase cells  were grown up on the day of the experiment in species-specific Hypho growth medium  was inoculated in each well; this avoids density-dependent confounding effects on phenotypic antibiotic resistance. Antibiotic stocks were prepared within 2 days of the experiment such that 2 μl of stock could be added to each well to achieve the desired gradient concentrations. Plates were then incubated at 30°C with shaking for 48 h. A Tecan plate reader was then used to measure the OD at 600 nm (OD600) and species-specific fluorescence (cyan fluorescent protein [CFP] for E. coli and yellow fluorescent protein [YFP] for S. enterica). The MIC90 was then used to establish which wells showed growth. Any well that had an OD600 or fluorescent protein value above 10% of the highest plate value was considered growth. We used the highest plate value rather than the antibiotic-free well because we consistently saw a slight increase in OD600 in the cocultures at sublethal concentrations, possibly due to a low level of cell lysis and a subsequent boost for the cross-feeding partner (We performed checkerboard assays (described below) with six antibiotics in 10 different combinations predicted to synergize (three combinations), antagonize (three combinations), or not interact (four combinations); see h medium  and 2 μl partner , 62.A =  and FICB = . FIC values were obtained for each well at the edge of growth, as shown in A and FICB , but not vice versa . In S. enterica, antibiotic A antagonizes antibiotic B but not vice versa. This leads to a “cancelling out” of the antagonistic interactions in coculture and causes the antibiotics to interact independently. In scenario 4, E. coli is the most susceptible species in both antibiotics. Therefore, the coculture antibiotic interaction pattern exactly matches that of E. coli. We then calculated FICs and FICIs for these predicted coculture plates and compared them to our observed data. We then used a Mann-Whitney U test to compare predicted versus observed FICIs for our cocultures, as we had insufficient sample sizes to determine whether our data were normally distributed (a necessary assumption for performing a t test).Based on observed monoculture growth patterns (MICs and FICs in each antibiotic combination), we predicted coculture growth patterns assuming weakest-link dynamics; that is, cocultures should only grow at concentrations of both antibiotics where both species are able to grow in monoculture. These predictions are outlined in