Antimicrobial resistance (AMR) is a global crisis with impacts on the future health and welfare of humans and animals. Determining key factors that influence veterinarians’ antimicrobial prescribing behaviours can bridge the gap between prescribing guidelines and clinical usage. Veterinarians practicing in Australia were surveyed on their frequency in prescribing different antibiotics; factors influencing their antibiotic prescribing behaviours; and their perceptions of current drivers of AMR. Antibiotics were prescribed in a third of consultations with key differences in the frequency of use of specific antibiotics by small companion animal (SCA), equine and livestock veterinarians, which broadly aligned with antibiotic registration restrictions in Australia. SCA veterinarians reported prescribing broad-spectrum antibiotics of higher importance to human health more frequently than livestock veterinarians. Factors that were reported as ‘strong’ or ‘moderate’ barriers to appropriate antibiotic prescribing were the 1) cost of culture and susceptibility testing and 2) lack of access to rapid and affordable diagnostic tests. Fear of losing clients, colleague pressure, and lack of their own understanding about antibiotics were considered to be ‘no’ or ‘somewhat’ of a barrier to appropriate prescribing by respondents. SCA veterinarians placed greater importance on the contribution of antibiotic use in livestock to AMR, than antibiotic use in companion animals. Despite reporting use of fewer, mostly narrow spectrum antibiotics of lower importance to human and animal health, livestock veterinarians were generally more aware of their potential contribution to AMR. This study provides insights into the similarities and differences in SCA, equine and livestock veterinarians practicing in Australia and informs sector-specific strategies to improve antimicrobial stewardship. Antimicrobial resistance (AMR) is a critical global health issue with potentially far-reaching impacts on the health of humans and animals . The gloFollowing the recommendations of the Swann report , AustralOur ability to capture the quantity and context of antimicrobial prescribing by veterinarians in Australia is currently limited to import quantities collated in periodic reports produced by the APVMA . Recent Globally, a range of both extrinsic and intrinsic factors—beyond scientific knowledge and clinical evidence—influence veterinarians. These include veterinarians’ antimicrobial preferences and professional experience; perceived antimicrobial efficacy; ease of patient administration; perceived owner compliance; animal characteristics; suboptimal housing conditions; biosecurity measures; cost of diagnostic tests; and social norms within clinical practices in Australia , the UniFactors influencing veterinarian prescribing behaviour and their perceptions of who contributes to the AMR issue needs to be better understood. We recently reported that prescribers of antibiotics tend to externalise the concept of who is most responsible for AMR, placing the contribution of other individuals  and professional sectors  before their own . Within Considering these priorities, our aim was to compare small companion animal (SCA), equine and livestock veterinarians with respect to their frequency in prescribing a range of different antibiotics; factors influencing their antibiotic prescribing behaviours; and their perceptions of current drivers of antimicrobial resistance.™ and distributed online between October and December 2016. Veterinary respondents were contacted via professional associations , state practitioner boards (required for veterinary registration) and other professional organisations using newsletters, bulletins, professional e-mail lists, forum announcements, social media sites and websites.Self-administered surveys were hosted on SurveyMonkeyA cross-sectional study was designed to target veterinarians registered in Australia across all sectors of the profession as part of a broader survey of antibiotic prescribing professionals in Australia  . Survey The survey comprised 45 questions: three open questions and 42 closed or semi-closed questions, mostly with Likert-type responses measured on four or five-point scales. “Unsure”, “neutral” and “not applicable (N/A)” options were also provided for specific questions, where appropriate. The survey was divided into four sections: information about the respondent and their practice; antibiotic prescribing behaviour and influences; information sources; and perceptions regarding antibiotics and antibiotic resistance. A copy of the survey is available as an online supplement  and was veterinarian-level characteristics including age, gender, years of veterinary practice experience (including year of graduation), place of graduation, and level of postgraduate education and whether the respondent had specialisation credentials; and b) practice-level characteristics including the type of veterinary practice , and practice location (postcode). To determine their antibiotic prescribing behaviour, respondents were asked to nominate the animal species they treated most frequently and to estimate the frequency with which they administered or dispensed a range of different antibiotics to this species from ‘never’ to ‘frequently’ . Responses to which animal species they most frequently treated were coded according to practice type as small companion animal , equine (horses) and livestock . Livestock were grouped together due to the absence of APMVA registration for many antibiotics of high importance in these species.Demographic data was collected at two levels: a) small companion animal’ , ‘equine’ (horse) and ‘livestock’  if the responding veterinarian spent more than 50% of their time treating animal species in that group. Those who did not indicate that they spent at least 50% of their time with any particular species were categorised according to the most common animal species they treated. Antibiotics were listed by their non-proprietary name, and if applicable, followed by a range of common veterinary drugs in that class, available in Australia. During analysis, the antibiotics were ranked as ‘high’, ‘medium’ or ‘low’ in accordance with the Australian Strategic and Technical Advisory Group (ASTAG) on importance rating for the mitigation of antibacterial resistance [For analysis of other questions  procedure, with a Bonferroni correction for multiple comparisons to estimate adjusted p-values. Kruskal-Wallis H statistical tests were also used to compare demographic sub-groups  within the group of veterinarians who mainly treated dogs (as this was the largest respondent group) as well as the relative frequency of this group’s prescription of different antibiotics for their patients. All “N/A” and “Unsure” responses were excluded from all statistical analyses. To compare mean antibiotic prescribing frequencies between the practice types, ANOVA was used. For all tests statistical significance was p < 0.05. The results of all analyses were collated . Medians and interquartile ranges (IQRs) were calculated and reported for questions with an ordinal response scale. Kruskal-Wallis H statistical tests were conducted to determine significant differences in median responses and mean ranks between different veterinarian groups; collated .Overall, responses were received from 403 veterinarians, representing 3.9% of the Australian national veterinary workforce. Of these responses, three veterinarians did not indicate which species they treated in their current or most recent role. Additionally, 13 respondents indicated they predominantly treated species  other than small companion animals (SCA), equine or livestock. Consequently, 16 responses were excluded from the statistical analysis. The remaining 387 responses were from veterinarians who spend most of their time treating SCA , horses  or livestock  . This isThose responding to the survey represented a range of ages and years of experience as practicing veterinarians, relative to the national workforce ,28. MoreRespondents reported a median case load of 40 patients per week (IQR: 50 patients). The reported median percentage of antibiotics prescribed / administered to patients per week was 33.3% (mean = 36.1%) with no significant difference found between practice types (p = 0.057). The predominant animal species that veterinary respondents treated in their clinical practice were dogs (n = 293), followed by horses (n = 26), cats (n = 23), cattle (n = 19), goats / sheep (n = 14), pigs (n = 5) and poultry (n = 2).Among SCA veterinarians, the antibiotics reported as most commonly prescribed / administered were amoxicillin-clavulanate (median–‘frequently’) followed by first generation cephalosporins , metronidazole, doxycycline / tetracycline, enrofloxacin and third generation cephalosporins (median–‘sometimes’) . ConsideFor the treatment of dogs, antibiotics that respondents used ‘frequently’ were amoxicillin-clavulanate and cephalexin / cephazolin . Those r‘Frequently’ prescribed antibiotics specifically for cats were amoxicillin-clavulanate and doxycycline . First gEquine veterinarians reported the most ‘frequently’ prescribed / administered antibiotics were trimethoprim-sulfamethoxazole, penicillin G and gentamicin; followed by tetracycline/doxycycline (median–‘sometimes’) . AntibioAmong livestock veterinarians, the frequency of antibiotic use was highly variable. Antibiotics reported as most commonly prescribed / administered were tetracycline / doxycycline , penicillin G , amoxicillin / ampicillin  and trimethoprim-sulfamethoxazole . The highest reported use of ceftiofur was ‘frequently’ . In general, for antibiotics of high importance to human and animal health, the median frequency of reported use was ‘never’, whereas for antibiotics of ‘medium’ or ‘low’ human health importance, the median frequency of use in livestock was up to ‘rarely’ or ‘sometimes’, respectively.Factors reported as ‘strongly’ or ‘moderately’ influencing antibiotic prescribing for all practice types were the patient’s clinical signs and medical history; the critical nature of patient illness; the patient’s antibiotic use history; the veterinarian’s experience in managing similar problems; culture and susceptibility testing results; patient safety; risk of promoting AMR in the patient or in the community; antibiotic potential for inducing adverse effects and to provide immediate patient pain relief . FactorsFactors that were a ‘significant’ or ‘moderate’ barrier to appropriate antibiotic prescribing for all practice types were the cost of culture and susceptibility testing and lack of rapid diagnostic tests . FactorsApproximately half of all SCA , equine , and livestock veterinarians  indicated that there were some antibiotics they do not feel comfortable prescribing or administering. These included fluoroquinolones (n = 88), third generation cephalosporins (n = 46), vancomycin (n = 19), chloramphenicol (n = 12) and carbapenems (n = 21). Reasons included the importance of these drugs in human medicine or recognition of these as last-line antibiotics (n = 24); need to ensure their use is supported by culture and susceptibility testing (n = 9) and concerns regarding promotion of antimicrobial resistance (n = 7).An additional factor generally rated as ‘strongly’ influencing selection of specific antibiotics by all practice types was the spectrum of antibiotic activity (broad vs narrow spectrum) . An addiThe majority of veterinarians perceived current levels of antibiotic use in human hospitals, nursing homes / aged-care facilities, general medical practice and unregulated use of antibiotics globally to be a ‘significant’ or ‘moderate’ contributor to the issue of antibiotic resistance, and a ‘significant’ problem for people in developed countries . LivestoPerceptions of the threat of antibiotic resistance to veterinary patients were more mixed between practice types but generally perceived as a problem. All practice type groups agreed that AMR was a ‘moderate’ problem in livestock and the food industry. Equine veterinarians considered AMR in horses, dogs and cats to be a ‘moderate’ problem whereas SCA and livestock veterinarians considered it to be a ‘minor’ problem. The majority of SCA (80.9%), equine (73.9%) and livestock (69.4%) veterinarians have seen antibiotic-resistant infections in patients and have experienced treatment failure with antibiotics. However, SCA veterinarians, and both equine and livestock veterinarians considered AMR to be a ‘minor’ and ‘moderate’ problem in their patients, respectively.All practice types agreed that ‘antibiotic use in minor or self-limited illnesses’, ‘too many prescriptions’ and ‘too low doses of antibiotics’ contributed ‘significantly’ to antimicrobial resistance. Veterinarians generally thought that patients / clients ‘not finishing prescribed course of antibiotics’, ‘not removing the site/source of infection’, prescribing an antibiotic when the benefit to the patient is uncertain’ and ‘using antibiotics from a previously unfinished course’ was a ‘moderate’ or ‘significant’ contributor to antibiotic resistance. All practice types generally agreed that ‘prescribing broad spectrum antibiotics when equally effective narrower spectrum antibiotics are available’; ‘too long duration of antibiotic treatment’ and ‘continuing antibiotics without appropriate laboratory investigation’ were a ‘significant’ to ‘moderate’ contributor to AMR. There was general agreement that environmental sources through environmental contamination with antibiotic waste, poor hand and healthcare hygiene, and transfer of resistant bacteria between humans, animals and the environment to be ‘moderate’ contributors to AMR.Key differences in the reported frequency of prescribing specific antibiotics by the three veterinarian groups were discovered in this study. There was commonality in factors influencing antibiotic prescribing behaviour; but there were some important disparities in the perception of respondents regarding current drivers of AMR. This information can be used when developing prescriber guidelines and sector-specific stewardship programs for Australian veterinarians.Current antimicrobial prescribing guidelines in veterinary practice focus mainly on clinical, microbiological and pharmacological indications for prescribing . The curSurvey respondents reported their clients’ or colleagues’ expectations had ‘no’ or ‘minimal’ influence despite a high proportion perceiving client pressure to prescribe. Using semi-structured interviews , HardefeThe cost of culture and antimicrobial susceptibility testing (AST); lack of rapid, affordable diagnostic tests for determining whether antibiotics are required; and identifying the most suitable antibiotic for patients’ infections, have been identified globally as a consistent barrier to the appropriate prescribing of antibiotics by veterinarians ,32. WhilThe frequency of prescribing specific antibiotics reported by veterinarians in this survey broadly reflected the availability and restrictions imposed in Australia by the APVMA on the registration of antibiotics for use in animals. The only antibiotic of critical importance registered for use in livestock in Australia is the third-generation cephalosporin ceftiofur, registered for use in cattle for bovine lower respiratory disease and single use for foot abscesses. Conversely a range of high importance antibiotics—such as the fluoroquinolone class —are registered for use in dogs and cats, while the third-generation cephalosporin ceftiofur is registered for use in dogs and horses, and cefovecin is registered for some soft tissue and urinary infections in dogs and cats in Australia. Under the legislation in the Stock Medicines Act governed by each state in Australia, veterinarians are permitted to use their professional judgement to prescribe antimicrobials off-label only for individual animals and not for conditions affecting animal herds or groups. While the concept of off-label use in veterinary practice has been criticised, current legislation restricts the updating of antimicrobial labelling to reflect current effective doses for common veterinary pathogens . RestricThe use of antibiotics in production animals, especially food-producing, has been the focus of an increasing number of studies –36 and mAntibiotics were reported to be dispensed in a third of consultations by Australian veterinarians across all practice types. While it is recognized that the self-reporting of antibiotic use and its frequency is a limitation of this study, it does provide broad trends on antibiotic use. In SCA veterinarians, this is comparable to reports from the UK, in which antibiotics were prescribed in 21% of feline and 25% of canine consultations in an analysis of medical records across 374 practices  and a lanarrow spectrum antibiotics were preferred over broad spectrum’ but this was not reflected in frequencies of use, with three of the top five antibiotics used being broad spectrum and of medium to high importance to human and animal health [The frequent use of the potentiated aminopenicillin amoxicillin-clavulanate in SCA reported in our study is a consistent and concerning finding in surveys of small animal practitioners globally ,41,42. Il health . Conversl health  and is al health .Rhodococcus equi, has been linked to the persistence of rifampicin-resistant ST612 methicillin-resistant Staphylococcus aureus in Australia and South Africa [The use of horses as both companion and working animals but classified as a food producing animal by APVMA has restricted the registration of certain antibiotics of high importance for this species in Australia, such as the fluoroquinolones. Trimethoprim-sulphonamides are the most frequently administered antibiotics by equine veterinarians, consistent with previous studies ,44 due th Africa –47. Equih Africa .Staphylococcus spp. and Escherichia coli, to determine the frequency of antimicrobial resistance in clinical isolates across Australian animal species. These have found higher frequencies of antibiotic resistance in clinical isolates from companion animals (including horses) than livestock species in these common bacterial species, with the frequency of fluoroquinolone resistance in companion animals comparable to human medicine, providing potential insights into the link between antimicrobial regulation, usage and resistance development. A national AMR surveillance network that reports on not only national veterinary clinical isolates, but the frequency of carriage of AMR within commensals and estimates the environmental impact of antibiotic use is required.Surveillance of AMR in microbial isolates from clinically normal and sick humans and animals provide essential links to the potential impact of antibiotic usage . AustralThis study confirms that across all veterinary practice types there is a need for affordable and rapid diagnostic testing, culture and AST to improve antimicrobial prescribing. Progress and compliance with antimicrobial stewardship principles is evident in livestock veterinarians whose use of critically important antibiotics was low and their awareness of their role in AMR was higher than SCA and equine veterinarians. The greatest disconnect between attitudes and practice was in SCA veterinarians. Access to national consensus antimicrobial guidelines, education on the importance rating of antimicrobial agents and the availability of narrow spectrum antibiotics of lower importance is essential for antimicrobial stewardship program success in all aspect of veterinary practice.S1 Survey(DOCX)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


International research networks have the potential to accelerate scientific progress via knowledge sharing and collaboration. In 2018, the US National Cancer Institute evaluated the International Cancer Screening Network (ICSN), in operation since 1988.ICSN hosts a biennial scientific meeting and scientific working groups. A survey was fielded to 665 ICSN participants, and a bibliometric analysis was conducted for ICSN publications.A total of 243 individuals completed the survey (36.5%). They reported that participating in the ICSN helped advance their knowledge of cancer screening research (75.7%), policy development (56%), and implementation (47.7%). Approximately three-quarters agreed that ICSN facilitated knowledge sharing and networking among researchers and implementers (79.9%) and those working on different continents (74.0%) and cancer sites (73.7%). More than half reported that participating helped them form new collaborations in screening implementation (58.0%) or research (57.6%). Most agreed that ICSN helped to advance screening research and evaluation (75.4%), effective screening practices (71.2%), and screening policies (60.9%). Many reported that participating informed advances in their own research (68.7%) and screening implementation (50.2%) and policies (49.4%) in their settings. Approximately two-thirds agreed that ICSN helped advance career development among current experts (66.6%) and train the next generation (62.2%). Half (51.4%) reported that participating advanced their own careers. The 20 ICSN publications included 75 coauthors. They were cited in 589 publications with more than 2,000 coauthors.Findings provide evidence of the influence of ICSN on international knowledge dissemination, collaboration, and advances in cancer screening research, implementation, and policies and highlight the potential value of longstanding international research networks. Through these approaches, these organizations are enhancing research rigor, enabling discoveries around rare events, and producing knowledge about the influence of multilevel interacting variables on health outcomes .7 However, these large research collaborations also require unique investments in the leadership, management, and conduct of the science—for example, leading development of a shared scientific vision and goals, facilitating communication, coordinating workflow, and establishing interoperability of data systems.8 These processes are critical to achieving scientific goals and also require additional investments of both financial and human resources.10Given these potential outcomes, new international networks and consortia continue to form, taking on new subjects and themes.It is important to evaluate the outcomes and impacts of networks and consortia to better understand the added value of these approaches, in light of the resources required for their successful implementation. In 2018 to 2019, the National Cancer Institute (NCI) of the US National Institutes of Health evaluated the International Cancer Screening Network (ICSN). An NCI-supported international research network, the ICSN has been in continuous operation since 1988, making it one of the longest-standing international research networks. Evaluation findings are reported here, and implications are discussed for understanding the value of longstanding international research networks.11 It pursues this aim by facilitating international knowledge sharing and research collaboration. The core activities of the ICSN are a biennial international scientific meeting and international scientific working groups. The ICSN is led and administered by the NCI. Biennial meetings and working groups are chaired by volunteer ICSN members.The ICSN aims to reduce the global burden of cancer by advancing research that improves the reach and effectiveness of cancer screening.11 Since then, the ICSN has grown into a global cancer screening research network. The 2019 biennial scientific meeting was attended by 311 individuals from 37 countries.The first ICSN meeting, held in 1988, was attended by an invited group of 24 cancer screening researchers from 11 countries who convened to discuss the potential for cross-national efforts to assess screening mammography diffusion and effectiveness.ICSN biennial meetings are open to the cancer screening community and provide a forum for scientific discussion and dissemination of the most current cancer screening research methods and findings. Content emphasizes evaluating the effectiveness of screening for cancer sites where screening has been documented to reduce mortality .ICSN scientific working groups identify key scientific priority areas in cancer screening and conduct international collaborative research on these topics. Currently active working groups are conducting research on international mammography screening skills, failures in the cervical cancer screening process, longitudinal adherence to fecal immunochemical testing for colorectal cancer screening, and overdiagnosis. Former ICSN working groups have conducted research on screening participation rates, health communication, performance evaluation, quality assurance, and other key topics. For a list of working group publications, see Appendix In 2018, the US NCI evaluated the ICSN to assess the outcomes and impacts of this long-running international research network. Research questions were:What are ICSN participants’ professional experiences and interests in the cancer screening field? andWhat have been the outcomes and impacts of the ICSN for:○ Participants’ knowledge of cancer screening research and implementation;○ Knowledge sharing, networking, and collaboration in the cancer screening field;○ Participants’ professional activities in cancer screening;○ Training and career development for the cancer screening community;○ Cancer screening research and evaluation; and○ Cancer screening implementation and policy?The study design included a survey of ICSN participants and a bibliometric analysis of ICSN-produced publications. The survey was fielded in the spring of 2018 to 665 individuals who had participated in the 2015 and/or 2017 ICSN biennial meetings and/or who had subscribed to the ICSN listserv. The survey invitation was sent via e-mail and included an embedded link to the online self-administered survey. To incentivize participation, five survey respondents were randomly selected to receive a US$100 gift card.The survey instrument included 43 questions (Data Supplement) in two sections. Section 1 addressed respondents’ professional experiences and interests in cancer screening and their attendance at ICSN meetings. Section 2 addressed the remaining research questions. The survey instrument was developed on the basis of formative interviews conducted by one of the coauthors (A.L.V.) with 14 current and former members of the ICSN Steering Committee. In addition, members of the ICSN Steering Committee and the ICSN directors at the NCI (coauthors S.H.T. and D.M.P.P.) reviewed drafts of the instrument. It was then pretested with global health professionals and finalized. Quantitative responses were analyzed in SAS  and qualitative responses were analyzed in Excel . The National Institutes of Health Office of Human Subjects Research Protections approved this study.12 The Science of Science Tool was used to analyze coauthor data, and Gephi was used to draw coauthorship networks.The bibliometric analysis addressed the impact of ICSN on international research collaboration and dissemination of knowledge in cancer screening. Bibliometric analyses examined coauthor country affiliations and coauthorship networks of ICSN-produced publications and citing publications. The base set of ICSN-produced publications was composed of articles attributed to the ICSN or an ICSN working group in the coauthor list or the acknowledgments section. Web of Science was used to identify citing articles through 2018. Income levels for coauthors’ countries were classified on the basis of the Atlas method defined by the World Bank.A total of 265 individuals completed the first part of the survey (39.8% response rate), and 243 individuals completed the full survey (36.5% response rate). Just under half had 10 or fewer years of experience in the cancer screening field (46.5%), and the remaining respondents had 11 or more years of experience . Most re12 Just more than one third worked primarily in Europe and Central Asia (39.6%) or North America (36.2%), respectively, and the remaining quarter (24.2%) were from the other five world regions combined. Approximately one third (32.5%) spent more than 25% of their work time focused on cancer screening in lower-resource settings in their home country or elsewhere. Approximately three quarters (72.5%) did most of their cancer screening work in the context of organized screening programs.When asked what country they worked in, primarily, respondents named 95 countries in all seven world regions.All respondents had participated in one or more ICSN biennial meetings. Most (83.0%) attended the 2015 and/or 2017 meeting. Approximately one quarter (26.4%) participated in the 2012 meeting, and approximately one third (32.5%) participated in prior meetings, where attendance was capped (2010) or by invitation only (2008 and earlier).Respondents were asked whether participating in the ICSN had helped to advance their knowledge of a range of professional activities in cancer screening . Three qResponses to an open-ended question about whether participating in the ICSN had advanced their knowledge in other topic areas highlighted four key topics: cancer risk, including risk prediction modeling; risks of screening, including overdiagnosis and adverse events; principles of and key issues in screening for specific cancer sites, including those within and outside of one’s area(s) of expertise; and knowledge of the range of screening practices internationally.Most respondents agreed that ICSN facilitated knowledge sharing and networking among diverse participants, including individuals engaged in cancer screening research and implementation (79.9%), living and working on different continents (74.0%), focused on different cancer sites (73.7%), and working in both high- and low-resource settings (62.2%). In addition, approximately three quarters (74.9%) of respondents agreed that ICSN has helped to form new international collaborations among cancer screening researchers, and approximately half (56.4%) agreed that ICSN had helped to form new international collaborations among cancer screening implementers .Most respondents also reported that participating in the ICSN produced benefits for them, personally, regarding knowledge sharing, networking, and collaboration . They reApproximately two-thirds of respondents agreed that ICSN has helped to advance the career development of current cancer screening experts (66.6%) and train the next generation of cancer screening experts (62.2%). In addition, approximately half of respondents (51.4%) reported that participating in the ICSN had advanced their own career development.Approximately three quarters of respondents agreed that ICSN has helped to advance screening research and evaluation (75.4%), and nearly as many respondents (68.7%) reported that their own participation in the ICSN had informed advances in screening research or evaluation approaches and/or methods they used in their own work .Focusing on screening implementation, most respondents agreed that the ICSN had helped to advance effective practices in cancer screening (71.2%) and cancer screening policies (60.9%). In addition, approximately half of respondents reported that their participation in the ICSN had informed advances in screening implementation (50.2%) and policies (49.4%) in their own work settings. In an overall assessment of the value of the ICSN, nearly three quarters of respondents agreed that the ICSN had helped to shape the field of cancer screening (73.6%).In open-ended comments, respondents attributed key successes of the ICSN—around facilitating knowledge acquisition, networking, collaboration, and advances in cancer screening research and implementation—in part to the design of the ICSN, including the network’s specialized focus on screening only, and the diversity of ICSN participants. In particular, they highlighted the value of including individuals from all over the world whose interests spanned cancer sites and whose professional activities, as a group, focused on both research and implementation. They also described how these characteristics enabled dissemination of effective policies and practices for cancer screening .Bibliometric analyses identified 20 publications produced by ICSN scientific working groups and the ICSN leadership group Appendix . These pThese 20 publications were cited in 589 other publications, which together had more than 2,000 coauthors located in 57 countries. These included 39 HICs and 18 low- and middle-income countries, of which 14 were upper-middle–income countries and four were lower-middle–income countries. Of these 589 citing publications, 16 were other ICSN publications. The coauthorship network diagram for ICSN publications depicts collaboration patterns among countries represented by coauthors of the 20 ICSN publications, in conjunction with country income categories . It highThe coauthorship network diagram for citing publications likewise highlights collaboration patterns, this time among countries represented among citing publication coauthors . It showThe current evaluation of the ICSN provided an opportunity to assess a 30-year-old international research group for participants, the science, and translational applications. In doing so, it addressed the question: what is the value of longstanding investments in international research collaboration?Findings reflect ICSN participants’ strong professional focus on cancer screening research, as expected given the research focus of the network . They alSurvey results documented the contributions of the ICSN to knowledge acquisition, with particularly strong agreement that ICSN advanced participants’ knowledge of conducting cancer screening research, conducting quality assurance, and engaging in policy development or advocacy . Topics There was strong agreement that ICSN facilitated knowledge sharing and networking across diverse participants and helped to form new international collaborations among screening researchers and implementers . This waThere was strong agreement that the ICSN has helped to advance cancer screening research and evaluation, shape the field of cancer screening, advance effective screening implementation, and advance effective screening policies . ReporteBibliometrics showed that the ICSN working groups and leadership group produced 20 publications. This modest number reflects the fact that publications are only one component of ICSN’s activities. In addition, many of these publications report on complex multinational comparative research requiring years to implement. The citing publication data show that ICSN publications, although few in number, have had an important influence on the field in both HICs and low- and middle-income countries . FurtherQualitative findings attributed the effectiveness of the ICSN to the combination of its specific focus on cancer screening and the diversity of participants’ professional experiences and knowledge within this field—across cancer screening sites, countries, high- and low-resource settings, and both research and implementation . ParticiA key limitation of this evaluation was that the sampling frame excluded individuals who have not attended ICSN, who may have different perceptions of the network’s value. In addition, bibliometrics documented, to some extent, the impact of the ICSN on the screening literature but cannot measure impact on cancer screening implementation. Finally, this evaluation lacked a comparison group.Future evaluations of other research networks and consortia might answer questions such as: How do these groups influence translational outcomes, and what outcomes do they influence? How do voluntary networks, funded networks, and funded portfolios of investigator-driven research compare in their influence on the science and translational applications? And how do varied approaches to the leadership, management, and administration of these groups, as well as varied scientific goals , influence outcomes?Recent scientific trends are amplifying the potential of international research consortia and networks to advance screening research. We are therefore likely to see an increase in these approaches. Evaluation of these initiatives is essential to fully understand their value. Findings from this evaluation suggest the potential for longstanding international research networks to contribute to knowledge sharing and collaboration and advancement of research methods and findings, related policies and practices, and fields of science.


Lactobacillus salivarius feeding could reverse antibiotic-induced lung defense impairment in a ventilator model.Widespread use of antibiotics in the intensive care unit is a potential cause of the emergence of hospital-acquired pneumonia. This study determined whether Lactobacillus salivarius and fructo-oligosaccharides (FOS) feeding were used to stimulate antibacterial protein expression in the intestine. Reactive oxygen species (ROS) in the intestinal mucosa was detected using 2ʹ7ʹ-dichlorofluorescein diacetate. The peroxynitrite production of alveolar macrophages (AMs) was measured using dihydrorhodamine 123 oxidation assay. N-acetylcysteine (NAC), an ROS scavenger, was orally administered to mice receiving antibiotics with FOS feeding.C57BL/6 wild-type (WT) mice received mechanical ventilation for 3 h after intramuscular antibiotic treatment for 6 days. Treatment with dead Pseudomonas aeruginosa (PA) phagocytic activity and activity of AMs and protein expression of regenerating islet-derived protein 3β (Reg3β) as well as Toll-like receptor 4 (TLR4) in the intestinal mucosa in the ventilator model. Antibiotic treatment also decreased ROS production in the intestinal mucosa, peroxynitrite production of AMs, and RELMβ expression as well as NF-κB DNA binding activity of the intestinal mucosa in WT mice but not in MyD88−/− mice. Treatment with dead L. salivarius or FOS feeding increased ROS production, bacterial killing activity, and protein expression of Reg3β as well as TLR4 in the intestinal mucosa and reversed the inhibitory effects of antibiotics on PA phagocytic activity of AMs.Antibiotic treatment decreased L. salivarius or FOS feeding reverses the antibiotic-induced lung defense impairment through the intestinal ROS/MyD88 pathways.Taken together with the finding that ablation of FOS-induced intestinal ROS using NAC decreased peroxynitrite production as well as PA phagocytic activity of AMs and protein expression of CRP-ductin, IL-17, Reg3β, and RELMβ in the intestinal mucosa, we conclude that commensal microflora plays a key role in stimulating lung immunity. Intestinal ROS plays a role as a predictive indicator and modulator of pulmonary defense mechanisms. Antibiotic treatment reduces lung defense against PA infection through the decrease in intestinal Reg3β and TLR4 expression. Treatment with dead  Pseudomonas aeruginosa (PA) is the most prevailing multidrug resistant (MDR) Gram-negative bacterium causing VAP [Ventilator-associated pneumonia (VAP) remain to be a serious complication in critical patients receiving mechanical ventilation (MV) for > 48 h . Pseudomsing VAP . It caussing VAP , 4. VAP sing VAP . The presing VAP , 7. HoweThe emergence of antibiotic-resistant bacteria that are difficult or impossible to treat is becoming more and more common and causing a global health crisis. More than 2 million people are infected with antibiotic-resistant bacteria with 23,000 deaths in the United States every year . ImpairmEnterobacteriaceae such as Klebsiella pneumoniae [K. pneumonia due to reduction in the mucosal bacterial killing activity and the expression of nondefensin proteins [L. salivarius is found in the mouth and the small intestine and is believed to play a role in enhancing the immune system [The human GI tract is colonized by trillions of microorganisms that include hundreds of various species of viruses and bacteria , 10. Theeumoniae . Moreoveeumoniae . Epitheleumoniae . The moseumoniae . Reg3β ieumoniae . Expresseumoniae . Howevereumoniae . We had proteins . Recentlproteins –21. The proteins . We hypoe system . The sec−/− (C57BL/6 background) mice were obtained from Oriental Bioservice, Inc . Experiments were in compliance with regulations approved by Kaohsiung Veterans General Hospital Animal Experiment Committee. All animal procedures were in compliance with the regulations on animals used for experimental and other scientific purposes approved by the Kaohsiung Veterans General Hospital Animal Experiments Committee.Specific pathogen-free (SPF) C57BL/6J mice were obtained from the National Laboratory Breeding and Research Center . Myd88To simulate the use of combined antibiotic treatment in clinical patients with infections, we used a broad-spectrum antibiotic treatment regimen in which animals received a combined intramuscular injection of different antibiotics as follows: ampicillin , vancomycin , and metronidazole , 100 μl/day . C57BL/6Lactobacillus or Bifidobacteria), mice were fed with dead Lactobacillus salivarius CECT5713 [8 CFU/ml) for 6 days. C57BL/6 mice were randomly divided into five groups as follows: Group I received intramuscular injection of normal saline for 6 days and sham operation; Group II received intramuscular injection of normal saline for 6 days and MV for 3 h; Group III received intramuscular injection of antibiotics for 6 days and MV for 3 h; Group IV received antibiotics injection with FOS feeding for 6 days and MV for 3 h; and Group V received antibiotics injection with dead Lactobacillus salivarius CECT5713 [8 CFU/ml) feeding for 6 days and antibiotics injection for 6 days and MV for 3 h. The ileum was harvested for western blotting. Phagocytic activity and TNF-α production of AMs were measured in another set of animals.To examine the effect of prebiotic feeding on antibiotic treatment-induced lung defense impairment in the ventilator model, prebiotic FOS (Sigma) were given to mice (250 mg/day) to stimulate the growth of probiotic bacteria for 6 days. To further prove that the mechanisms for improvement and prevention by FOS supplementation are through increasing specific groups of intestinal commensal microbiota  were given to mice for 6 days. C57BL/6 mice were randomly divided into four groups as follows: Group I received intramuscular injection of normal saline for 6 days; Group II received intramuscular injection of antibiotics for 6 days; Group III received intramuscular injection of antibiotics and FOS feeding for 6 days; and Group IV received intramuscular injection of antibiotics and dead Lactobacillus salivarius CECT5713 [8 CFU/ml) feeding for 6 days. ROS production of the intestine and peroxynitrite production of AMs were examined. Another set of animals received PA intratracheal instillation. Animals were sacrificed at 8 h after PA instillation and lungs were harvested for myeloperoxidase activity assay.To investigate the association between ROS production in the intestine and PA-induced neutrophil infiltration in the lung, FOS (250 mg/day) or dead CECT5713  (2 × 108CECT5713  (2 × 108N-acetylcysteine (NAC), an ROS scavenger, was orally administered to mice receiving antibiotics with FOS treatment for 3 days.To examine the role of intestinal ROS production in antibiotic treatment-induced lung defense impairment; 0.1 mM C57BL/6 mice were randomly divided into four groups as follows: Group I received intramuscular injection of normal saline for 6 days; Group II received intramuscular injection of antibiotics for 6 days; Group III received intramuscular injection of antibiotics and FOS feeding for 6 days; and Group IV received intramuscular injection of antibiotics with FOS feeding for 6 days and NAC feeding for 3 days. The production of 2′7′-dichlorofluorescein diacetate (DCFDA) and antibacterial protein expression in the ileum and phagocytic activity and peroxynitrite production of AMs were measured in all animals.L. salivarius  [L. salivarius.To examine the effects of intestinal dysbiosis on lung defense mechanism, prebiotic  or dead  CFU/ml)  was suppMice were anesthetized with Avertin , and the neck was opened at 1 cm below the mouth. Muscles were divided and the trachea was cut and cannulated with 0.7 cm 21G flat syringe needle that connected to a mechanical ventilator  for 3 h. During the duration of mechanical ventilation, the mice were administered Avertin (15 mg/kg) and sterile saline (10 μl/g) every hour. The ventilation protocol was high stretch  and without end expiratory pressure (PEEP). The control mice breathed spontaneously during 3-h period.g, 4 °C for 10 min. Supernatants (60 μl) was added to 939 μl of potassium phosphate buffer with 16.7 mg/ml of O-dianisidine and 0.5% hydrogen peroxide. The rate of change in absorbance at 460 nm was examined over 2 min. One unit of MPO activity is determined as the amount of enzyme that reduces 1 μmol of peroxide per min.Lung myeloperoxidase (MPO) activity is a marker of lung neutrophil infiltration . Lung tiP. aeruginosa and epithelial cells promote the neutrophil sequestration into lungs which may cause lung injury as well as active the host immunity response [6 cells/ml. After 5 min of preincubation, the cell suspension was incubated with P. aeruginosa (108/ml) at 37 °C for 1 h with shaking. The cells were removed as the pellet after centrifugation at 200×g for 10 min, and P. aeruginosa number in the supernatant was counted [Alveolar macrophages (AMs) represent the first line of lung defense against pathogens such as response . The alv counted , 28.5 cells/ml. Cells were then cultured in 96-well microtiter plates for 2 h and washed with RPMI 1640 to remove non adherent cells [Escherichia coli O26:B6 Sigma-Aldrich) or RPMI 1640 for 4 h. Supernatants were collected and stored at − 70 °C until assayed for TNF-α.AMs were harvested from adult mice by bronchoalveolar lavage (BAL) with Tris-buffered saline containing 0.25 mM EDTA and EGTA. AMs were resuspended in RPMI 1640 in a final concentration of 1 × 10nt cells . AdherenR&D Systems); the TLR4 were identified by mouse monoclonal, rabbit polyclonal and goat polyclonal antibodies, respectively (Santa Cruz Biotechnology Inc.).The Reg3β, RELMβ, IL17, and CRP-ductin were identified by mouse monoclonal antibodies (7 CFU P. aeruginosa) were instilled via an angiocatheter through the trachea.Mice were anesthetized with xylazine  and ketamine hydrochloride . The trachea was surgically opened and 50 µl , which was added into the suspension of intestinal mucosa for cultivation. DCFDA is deacetylated by cellular esterases to a non-fluorescent compound, which is oxidized by ROS into 2′7′-dichlorofluorescein (DCF). DCF is detected by fluorescence spectroscopy with excitation and emission spectra of 495 and 529 nm, respectively .6 ml−1 in RPMI + 10% FBS, and 100 μl of HBSS without phenol red containing 25 μM of 1,2,3-dihydrorhodamine , a peroxynitrite-detecting dye was added. The cells were stimulated with 1 μg/ml Escherichia coli LPS (Sigma). Peroxynitrite was determined using a fluorescence plate reader  every 15 min for 75 min using excitation and emission wavelengths of 485 and 530 nm, respectively. Fluorescence due to auto-oxidation of 123-DHR was subtracted from the original data [Peroxynitrite is a potent macrophage-derived oxidizing cytotoxin that attacks invading pathogens. The collected AMs were adjusted to 3.0 × 10nal data .All data were analyzed by one-way analysis of variance or T-test analysis of variance (ANOVA), followed by Turkey’s Multiple Comparison Test. All values in the figures and text were expressed as mean ± standard error of the mean. P values of less than 0.05 were considered to be statistically significant.To study the effects of MV and antibiotics on lung defense mechanisms, AMs of WT mice were harvested for PA phagocytic activity assay after different treatments. MV significantly decreased PA phagocytic activity of AMs by 28% compared to that in the control group (40.27 ± 3.76 vs. 55.13 ± 1.96)         Fig. b. These L. salivarius feeding reversed the stimulatory effect of antibiotic treatment on PA pneumonia-induced neutrophil infiltration in the lungs compared to that in the antibiotic treatment group (13.42 ± 2.04 and 12.31 ± 0.43 vs. 27.76 ± 1.15 unit/g)    (Fig. /−− mice were examined. Antibiotic treatment decrease the DCFDA levels in the intestinal mucosa in WT mice but not in MyD88/−− mice. Moreover, FOS feeding after antibiotic treatment did not change DCFDA levels in the intestinal mucosa in MyD88/−− mice compared to those in the antibiotic treatment group (Fig. To examine the role of MyD88 in antibiotic-induced reduction of intestinal ROS production, DCFDA levels in the intestinal mucosa of MyD88oup Fig. a. These /−− mice was examined. Antibiotic treatment did not change the peroxynitrite production of AMs in MyD88/−− mice compared to that in the control group. Furthermore, FOS treatment after antibiotic treatment did not change the peroxynitrite production of AMs in MyD88/−− mice compared to that in the antibiotic treatment group (Fig. To examine the involvement of MyD88 in the inhibitory effect of antibiotic treatment on the lung defense mechanisms, peroxynitrite production of AMs in MyD88oup Fig. b. These /−− mice was examined. Antibiotic treatment did not change PA phagocytic activity of AMs in MyD88/−− mice compared to that in the control group. Furthermore, FOS treatment after antibiotic treatment did not change PA phagocytic activity of AMs in MyD88/−− mice compared to that in the antibiotic treatment group (Fig. To examine the role of MyD88 in the inhibitory effect of antibiotic treatment on the lung defense mechanisms, PA phagocytic activity of AMs in MyD88oup Fig. c. These /−− mice compared to that in the control group. Furthermore, FOS treatment after antibiotic treatment did not change the expression of Reg3β and RELMβ in MyD88/−− mice compared to that in the antibiotic treatment group (Fig. To examine the role of MyD88 in the inhibitory effect of antibiotic treatment on antibacterial protein expression in the intestinal mucosa, the protein expression of Reg3β and RELMβ in the intestinal mucosa was examined. Antibiotic treatment did not change the expression of Reg3β and RELMβ in the intestinal mucosa in MyD88oup Fig. d. These −/− mice compared to that in the control group. Furthermore, FOS treatment after antibiotic treatment did not change NF-κB DNA binding activity in MyD88/−− mice compared to that in the antibiotic treatment group (Fig. Antibiotic treatment did not change NF-κB DNA binding activity of the intestinal mucosa in MyD88oup Fig. e. These VAP is the main cause of nosocomial infection in the ICU and a risk factor for increased mortality. MV has been known to decrease lung defense mechanisms through the increase of ROS production of AMs . MV alsoL. salivarius to healthy adults has been proved to be safe and improve gut microbiota and different parameters related to immune response [L. salivarius, we fed antibiotic treatment animals with dead L. salivarius instead of living L. salivarius to enhance the immune system of intestinal tract. Previously, living L. salivarius have been proved to provide anti-inflammatory effect on TNBS-induced colonic damage in a rat model but not the dead strain [L. salivarius restored the protein expression of Reg3β and TLR4 in the intestinal mucosa and reversed the antibiotic-induced reduction of PA phagocytic activity of AMs. Therefore, we speculate that living L. salivarius will provide better enhancing effects on host defense mechanisms in the antibiotic treatment before MV model than the dead strain.Daily administration of living response . To decrd strain . Our datLactobacillus rhamnosus administration is safe in critically ill patients and efficacious for the prevention of VAP [L. salivarius. We clearly demonstrated that the gut microbiota plays a protective role in PA-induced VAP as evidenced by the decrease in the phagocytic activity and activity of AMs in mice treated with antibiotics. Oral feeding with FOS or dead L. salivarius in mice receiving antibiotic treatment before MV significantly increased the PA phagocytic activity of AMs. These results indicate that FOS or dead L. salivarius feeding reverses the inhibitory effects of antibiotic treatment on lung defense mechanism in the mice ventilator model. To examine the involvement of intestinal TLRs as well as antibacterial protein expression in the stimulatory effects of FOS or dead L. salivarius feeding on antibiotic-induced lung defense impairment, the protein expression of Reg3β and TLR4 in the intestinal mucosa was examined. Antibiotic treatment significantly decreased the protein expression of Reg3β and TLR4 in the intestinal mucosa compared to that in the MV group. FOS or dead L. salivarius feeding in mice receiving MV after antibiotic treatment significantly increased the protein expression of Reg3β and TLR4 in the intestinal mucosa compared to that in the antibiotic treatment before MV group. Together, our data suggest that antibiotic treatment decreases the protein expression of Reg3β and TLR4 in the intestinal mucosa in the mice ventilator model. Oral feeding with FOS or dead L. salivarius restores the protein expression of Reg3β and TLR4 in the intestinal mucosa and reverses the antibiotic-induced reduction of PA phagocytic activity of AMs. Our studies provide a basis for the development of a novel therapeutic strategy for VAP.Intestinal microbes not only contribute to the local defense against infections, but also modulate systemic immune response especially the lung . Recent n of VAP . To asseL. salivarius feeding in mice receiving antibiotic treatment significantly increased the DCFDA levels in the intestinal mucosa. These results indicate that antibiotic treatment decreases intestinal ROS levels and FOS or dead L. salivarius feeding restores them. To examine the mechanisms of antibiotic treatment in the lung defense, we further examined the peroxynitrite production of AMs. MV has been previously shown to decrease lung defense mechanisms through the increase of ROS production of AMs [L. salivarius in mice receiving antibiotic treatment significantly increased the peroxynitrite production of AMs compared to that in mice treated with antibiotics. These results indicate that antibiotic treatment decreases lung defense of AMs and FOS or dead L. salivarius feeding restores it. Furthermore, our data demonstrated that antibiotic treatment before PA intratracheal injection significantly increased PA pneumonia-induced neutrophil infiltration and FOS or dead L. salivarius feeding reversed the stimulatory effect of antibiotic treatment on PA pneumonia-induced neutrophil infiltration in the lungs. To further examine the involvement of intestinal ROS production in lung defense mechanisms, NAC, a ROS scavenger, was orally given to mice after FOS treatment. The stimulatory effect of FOS feeding on ROS production in the intestinal mucosa was abolished by NAC treatment compared to that in the antibiotic + FOS treatment group. The stimulatory effect of FOS feeding after antibiotic treatment on peroxynitrite production and the PA phagocytic activity of AMs were also abolished by NAC treatment. In summary, antibiotic treatment induces lung defense mechanism impairment through the decrease of ROS production of the gut mucosa, the peroxynitrite production of AMs, and the subsequent phagocytic activity of AMs. These results indicate that oral feeding with the intestinal ROS scavenger inhibits the stimulatory effect of FOS feeding on the lung defense mechanism by decreasing the intestinal ROS production and peroxynitrite production of AMs. Our data identify a gut–lung axis in VAP and establish a mechanism for pulmonary immunomodulation by the intestinal mucosa.ROS levels in the gut have been suggested to be closely related to the intestinal barrier function . Out datn of AMs . Our datL. salivarius feeding in mice receiving MV after antibiotic treatment significantly increased the protein expression of Reg3β and TLR4 in the intestinal mucosa compared to that in the antibiotic treatment with MV group. NAC treatment after FOS feeding significantly decreases FOS-induced CRP-ductin, IL-17, Reg3β, and RELMβ expression in the mucosa. NAC treatment could abolish the stimulatory effect of ROS on intestinal antibacterial protein expression and peroxynitrite production of AMs. These results suggest that ROS production in the intestinal mucosa plays an important role in the stimulatory effect of FOS feeding on the antibacterial protein expression in the intestinal mucosa.We further examine the role of intestinal antibacterial protein in the stimulatory effect of FOS treatment on lung defense mechanism. Antibiotic treatment significantly decreased the protein expression of CRP-ductin, IL-17, Reg3β, and RELMβ in the intestinal mucosa. FOS or dead /−− mice. Moreover, antibiotic treatment did not decrease Reg3β and RELMβ protein expression in the intestinal mucosa in MyD88/−− mice. Furthermore, FOS treatment after antibiotic treatment did not increase DCFDA levels and intestinal Reg3β as well as RELMβ protein expression, peroxynitrite production of AMs, and PA phagocytic activity of AMs in MyD88/−− mice. Altogether, these results indicate that antibiotic treatment decreases DCFDA levels in the intestinal mucosa, peroxynitrite production of AMs, and PA phagocytic activity of AMs through the MyD88 signaling pathway.Commensal microflora has been known to maintain systemic defense mechanisms. An earlier study showed that MyD88 is an important signaling pathway between bacteria and host defense mechanism . TherefoAntibiotic treatment decreases the protein expression of TLR4, CRP-ductin, IL-17, Reg3β, and RELMβ and ROS production in the intestinal mucosa. The decrease in ROS production in the intestinal mucosa induces the decrease in peroxynitrite production as well as phagocytic activity of AMs. FOS or dLac feeding increase ROS production and stimulates the protein expression of TLR4, CRP-ductin, IL-17, Reg3β, and RELMβ in the intestinal mucosa and peroxynitrite production as well as phagocytic activity of AMs. FOS treatment reverses the antibiotic-induced lung defense mechanism through MyD88 signaling. Commensal microflora plays an important role in stimulating lung defense through the induction of intestinal ROS production. These observations imply that oral feeding with a TLR4 stimulator may be useful to increase the lung defense mechanism in patients undergoing MV with antibiotic treatment.


Aedes aegypti larvae and adults and two field caught mosquito species Coquillettidia perturbans and Ochlerotatus canadensis. The cultured bacterial populations were dominated by isolates belonging to the class Gammaproteobacteria. Among the antibiotic resistant populations, we found bacteria resistant to carbenicillin, kanamycin, and tetracycline, including bacteria resistant to a cocktail of all three antibiotics in combination. The antibiotic resistant bacteria were numerically rare, at most 5% of total cell counts. Isolates were characterized by 16S rRNA gene sequencing, and clustering into Operational Taxonomic Units . 27 antibiotic resistant OTUs were identified, although members of an OTU did not always share the same resistance profile. This suggests the clustering was either not sensitive enough to distinguish different bacteria taxa or different antibiotic resistant sub-populations exist within an OTU. Finally, the antibiotic selection opened up a niche to culture mosquito-associated fungi, and 10 fungal OTUs (28S rRNA gene sequencing) were identified. Two fungal OTUs both classified to the class Microbotryomycetes were commonly identified in the field-caught mosquitoes. Thus, in this study we demonstrate that antibiotic resistant bacteria and certain fungi are common and conserved mosquito microbiome members. These observations highlight the potential of invertebrates to serve as vehicles for the spread of antibiotic resistance throughout the environment.The emerging and increasing prevalence of bacterial antibiotic resistance is a significant public health challenge. To begin to tackle this problem, it will be critical to not only understand the origins of this resistance but also document environmental reservoirs of antibiotic resistance. In this study we investigated the possibility that both colony and field caught mosquitoes could harbor antibiotic resistant bacteria. Specifically, we characterized the antibiotic resistant bacterial populations from colony-reared  Mosquitoes, like many complex organisms, live in association with a microbiome made up of bacteria, fungi and viruses e.g. –5. DescrThe generation of axenic mosquitoes, allowing for establishing gnotobiotic hosts with a controlled microbiome, has only been reported recently ,10. PrevIn this study we set out to test how common antibiotic resistant bacteria are among colony-reared and field-caught mosquitoes. We hypothesized that the mosquito microbiome would naturally harbor antibiotic resistant bacterial populations. We further predicted that the wild-caught mosquitoes would harbor larger and more diverse antibiotic resistant populations due to their increased probability of encountering antibiotics in their environment, as the colony mosquitoes had no history of antibiotic treatment.Aedes aegypti; Orlando strain) were maintained using standard conditions Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/AReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: This reviewer enjoyed very much in reading this excellent manuscript. This work combines the fields of the vector-borne diseases, antimicrobial resistance and microbiology. The manuscript is well written, and the conclusion is well supported by the data. Main comments:1) I strongly feel like that the portion of commensal fungi can be left out for another independent publication. Please focus on the beauty of this work, antimicrobial resistance and mosquitoes. Do not get distracted by the addition of fungi, in which you did not get any isolates, and cannot characterize the nature of their antimicrobial resistance;2) For the field-caught mosquitoes, did you try to wash the mosquitoes before performing bacterial isolation etc? There are constant contact between mosquitoes and animal hosts/environmental surfaces etc. The identified microbiome could be on the surface of the mosquitoes, or their inside? Please provide more information, and make meaningful discussion;3) As indicated in Fig 2 , you used LB initially, followed by the utilization of different selective media. Please justify this approach. What would you get if you would use the selective media directly without the use of LB media? Even LB is not a type of selective media, the process of LB culture may still provide some preference of certain bacteria which overgrow over others;4) It is great that the authors used the bioinformatics to identify bacterial OUTs in this work. In the meantime, it would be more clinically more important and meaningful if you could identify the genus level and even species level of the isolated bacterial from the antibiotics-resistant plates. You are very close to provide this valuable information since these isolates are available to you, and you can even ID them by the use of the whole-length 16S rRNA sequencing.Reviewer #2: Well done paper.On line 95 I think you are missing the word resistant - it states antibiotic populations and I think you meant to say antibiotic resistant populations. That is the only change that I have.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  18 Jul 2019Reviewer #1: This reviewer enjoyed very much in reading this excellent manuscript. This work combines the fields of the vector-borne diseases, antimicrobial resistance and microbiology. The manuscript is well written, and the conclusion is well supported by the data. Response:We would like to thank the reviewer for their kind comments. Main comments:1) I strongly feel like that the portion of commensal fungi can be left out for another independent publication. Please focus on the beauty of this work, antimicrobial resistance and mosquitoes. Do not get distracted by the addition of fungi, in which you did not get any isolates, and cannot characterize the nature of their antimicrobial resistance;Response:We feel that the fungal data should remain in the current manuscript. Our reasoning is as follows:These data are based on isolates. As we state in line 356 “through our efforts to culture antibiotic resistant bacteria we opened up a niche to culture fungal isolates from the mosquitoes”. We hypothesize that we recovered these fungal isolated by reducing the bacterial load on the antibiotic plates. We did not actually set out to culture fungi. In this regard our base media (LB) was not optimized for fungal recovery, as such the identification of fungi was a spurious finding in this study, not an objective. This data would not likely be sufficient for an independent manuscript. 2) For the field-caught mosquitoes, did you try to wash the mosquitoes before performing bacterial isolation etc? There are constant contact between mosquitoes and animal hosts/environmental surfaces etc. The identified microbiome could be on the surface of the mosquitoes, or their inside? Please provide more information, and make meaningful discussion;Response:This is something that we took care to control for but failed to mention in the methods. We have revised the methods to read “Prior to culturing individuals were washed in a solution of 90% ethanol to remove external adhering bacteria.” line 1483) As indicated in Fig 2 , you used LB initially, followed by the utilization of different selective media. Please justify this approach. What would you get if you would use the selective media directly without the use of LB media? Even LB is not a type of selective media, the process of LB culture may still provide some preference of certain bacteria which overgrow over others;Response:The selective plates were LB plates supplemented with antibiotics. The purpose of this was to determine the proportion of antibiotic resistant cells in the “total” population. To avoid confusion we have added “LB media supplemented with antibiotics” to the figure legend of Figure 2 to make it clear that the media formulation is the same between all of the plates. We agree with the reviewer that only using LB media likely influenced the bacteria that we recovered. We have added the following “However, as all culturing was performed on LB media these data likely only represent a proportion of the total bacterial diversity and favor fast growing bacterial species” to the results.4) It is great that the authors used the bioinformatics to identify bacterial OUTs in this work. In the meantime, it would be more clinically more important and meaningful if you could identify the genus level and even species level of the isolated bacterial from the antibiotics-resistant plates. You are very close to provide this valuable information since these isolates are available to you, and you can even ID them by the use of the whole-length 16S rRNA sequencing.Response:While we agree with the reviewer that full-length sequencing could improve some of the ability to discern OTU’s, we argue that performing full length sequencing is not warranted for the following reasons.1. Even full-length 16S rRNA gene sequencing is not generally considered to be sufficient to differentiate species or strains. This level of discrimination is generally performed by multi-locus sequencing, DNA-DNA reassociation kinetics, or whole genome sequencing, which is beyond the scope of this study.2. Even with the short fragment that we sequenced ~75% of the isolates were classified to genus with >80% confidence (line 232). In this respect, there is likely to be little improvement in our data with full-length gene sequencing.3. We have dedicated a section of our discussion (line 356) to discuss the potential limitations of using a short sequence fragment, so have already addressed this concern in the manuscript. Reviewer #2: Well done paper.On line 95 I think you are missing the word resistant - it states antibiotic populations and I think you meant to say antibiotic resistant populations. That is the only change that I have.Response:The suggested change has been made.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 1 Aug 2019Antibiotic resistant bacteria and commensal fungi are common and conserved in the mosquito microbiomePONE-D-19-16438R1Dear Dr. Steven,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact With kind regards,Patrick Butaye, DVM, PhDAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 5 Aug 2019PONE-D-19-16438R1 Antibiotic resistant bacteria and commensal fungi are common and conserved in the mosquito microbiome Dear Dr. Steven:I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. For any other questions or concerns, please email Thank you for submitting your work to PLOS ONE.With kind regards,PLOS ONE Editorial Office Staffon behalf ofProfessor Patrick Butaye Academic EditorPLOS ONE


Three types of polyhedral oligomeric silsesquioxanes (POSSs) with different functional active groups were used to modify rigid polyurethane foams (RPUFs). Aminopropylisobutyl-POSS (AP-POSS), trisilanoisobutyl-POSS (TS-POSS) and octa(3-hydroxy-3-methylbutyldimethylsiloxy-POSS (OH-POSS) were added in an amount of 0.5 wt.% of the polyol weight. The characteristics of fillers including the size of particles, evaluation of the dispersion of particles and their effect on the viscosity of the polyol premixes were performed. Next, the obtained foams were evaluated by their processing parameters, morphology , mechanical properties , viscoelastic behavior , thermal properties  and application properties . The results showed that the morphology of modified foams is significantly affected by the fillers typology, which resulted in inhomogeneous, irregular, large cell shapes and further affected the physical and mechanical properties of the resulting materials. RPUFs modified with AP-POSS represent better mechanical properties compared to the RPUFs modified with other POSS. The smallest values of thermal conductivity are observed for RPUFs with closed-cell structure, the apparent density of which is from 25 to 35 kg m−3 [Polyurethanes (PUs) are a group of polymers characterized by the most diverse properties, thanks to which they have a very wide range of industrial applications ,2,3. For5 kg m−3 . A signi5 kg m−3 ,16,17,18A review of the related literature indicates that one of the method of increased flammability of PU foams are fire-resistant coatings. For example, a layer-by layer assembly technique of polyethylenimine (PEI), graphene oxide (GO) and synthetic melanin nanoparticles (SMNPs) was constructed on the surface of PU foams . ObtaineIt was reported that, in addition to traditional fillers, an interesting group of reactive nanofillers are polyhedral oligomeric silsesquioxanes (POSS), which combine the features of organic and inorganic materials ,30,31. THybrid nature and nanometric size scale make silsesquioxanes extremely attractive materials used as nanofillers . It was The aim of this research is to develop and test PU foams modified with three type of POSS—two of them with closed-cage functionalized by amino- or hydroxyl group (Aminopropylisobutyl-POSS (AP-POSS) and octa(3-hydroxy-3-methylbutyldimethylsiloxy-POSS (OH-POSS) and one with open-cage (trisilanoisobutyl-POSS (TS-POSS)). The influence of the different type of POSSs on thermal properties , dynamic mechanical properties , physico-mechanical properties  and morphology of obtained PU composites was examined in current work. The obtained results, indicate that the addition of POSS influences the morphology of analyzed foams and consequently their further mechanical and thermal properties.N,N-Dimethylcyclohexylamine), flame retardant (Tris(2-chloro-1-methylethyl)phosphate), chain extender  and water as blowing agent [The water-blown PUR foams used in this study were obtained from a two-component system supplied by Purinova Sp. z o. o. from Bydgoszcz, Poland, after mixing the polyol (Izopianol 30/10/C) and the diphenylmethane diisocyanate (Purocyn B). The polyol is a mixture of components containing polyester polyol , catalyst (ng agent . This foCharacterization techniques) and their physico-mechanical properties were investigated. A schematic figure of the synthesis of RPUFs is presented in RPUFs containing 0.5 wt.% of AP-POSS, TS-POSS, and OH-POSS were obtained as follows. The adequate amount of selected POSS was added to the Izopianol 30/10/C and the mixture (component A) was homogenized with an overhead stirrer at 600 RPM under ambient conditions for approximately 60 s. Purocyn B (component B) was added to component A and the mixture was stirred for 10 s at a speed rate of 1800 RPM. Following the information provided by the supplier, the ingredients were mixed in the ratio of 100:160 (ratio of component A to component B). Such prepared system was poured into an open mold and allowed to expand freely in the vertical direction. RPUFs were conditioned at room temperature for 24 h. After this time, samples were cut with a band saw into appropriate shapes . The size of particles polyol dispersion (0.04 g/L) was determined with the dynamic light scattering DLS method.−1 at room temperature.The absolute viscosities of polyol and isocyanate were determined corresponding to ASTM D2930  using a rotary Viscometer DVII+ . The torque of samples was measured as a range of shear rate from 0.5 to 100 sThe apparent density of foams was determined accordingly to ASTM D1622 . The densities of five specimens per sample were measured and averaged.The morphology and cell size distribution of foams were examined from the cellular structure images of foam which were taken using JEOL JSM-5500 LV scanning electron microscopy . All microscopic observations were made in the high-vacuum mode and at the accelerating voltage of 10 kV. The samples were scanned in the free-rising direction. The average pore diameters, walls thickness, and pore size distribution were calculated using ImageJ software (Media Cybernetics Inc.).−1 up to 600 °C with the sample mass about 10 mg. The decomposition temperatures  were determined.The thermal properties of the synthesized composites were evaluated by TGA measurements performed using a STA 449 F1 Jupiter Analyzer . About 10 mg of the sample was placed in the TG pan and heated in an argon atmosphere at a rate of 10 K min10%σ) of foams was determined accordingly to the ASTM D1621  using Zwick Z100 Testing Machine  with a load cell of 2 kN and the speed of 2 mm min−1. Samples of the specified sizes were cut with a band saw in a direction perpendicular to the foam growth direction. Then, the analyzed sample was placed between two plates and the compression strength was measured as a ratio of the load causing 10% deformation of sample cross-section in the parallel and perpendicular direction to the square surface. The result was averaged of 5 measurements per each sample.The compressive strength  results for each sample were expressed as a mean value. The average of 5 measurements per each type of composition was accepted.Three-point bending test was carried out using Zwick Z100 Testing Machine  at room temperature, according to ASTM D7264 . The tested samples were bent with testing speed 2 mm min−1, using a frequency of 1 Hz and applied deformation at 0.1%.Dynamic mechanical analysis (DMA) was determined using ARES Rheometer . Torsion geometry was used with samples of thickness 2 mm. Measurements were examined in the temperature range 20–250 °C at a heating rate of 10 °C minSurface hydrophobicity was analyzed by contact angle measurements using the sessile-drop method with a manual contact angle goniometer with an optical system OS-45D  to capture the profile of a pure liquid on a solid substrate. A water drop of 1 μL was deposited onto the surface using a micrometer syringe fitted with a stainless steel needle. The contact angles reported are the average of at least ten tests on the same sample.Water absorption of the RPUFs was measured according to ASTM D2842 . Samples were dried for 1 h at 80 °C and then weighed. The samples were immersed in distilled water to a depth of 1 cm for 24 h. Afterward, the samples were removed from the water, held vertically for 10 s, the pendant drop was removed and then blotted between dry filter paper  at 10 s and weighed again. The average of 5 specimens was used.ol is the length of the sample before thermostating and l is the length of the sample after thermostating. The average of 5 measurements per each type of composition was reported.Changes in the linear dimensions were determined in accordance to the ASTM D2126 . The samples were conditioned at the temperature of 70 °C and −20 °C for 14 days. Change in linear dimensions was calculated in % from Equation (1).One of the most important parameters determining the behavior of the filler in the polymer matrix is the size of its particles. If the particles are too small, their dispersion may be difficult because they have a greater tendency to aggregate and agglomerate, forming large clusters in the matrix. Too large particles may affect the foaming process and further properties of the obtained materials. The particle size of the POSS powder was measured in a polyol dispersion (0.04 g/L). The results of particle size measurements are given in From the diagram, it follows that the size of AP-POSS particles ranges from 65 to 104 nm, the highest percentage—29% is shown by 82 nm particles. In the case of TS-POSS, the particle size distribution is somewhat larger and ranges from 59 to 108 nm, with the largest 69 nm volume fraction. Such small particle sizes of nanofillers may suggest their tendency to agglomerate in the polyol, which may negatively affect mechanical and functional properties. The viscosity of the reactive mixture was measured first since it is a critical parameter affecting the foaming process . Increasn) was calculated from the slopes. All results are presented in The rheological properties of polyol premixes are shown as the viscosity versus shear rate in maxT during the foaming process in each case. The presence of the additional groups as a result of the incorporation of the filler can lead to the exothermic reaction providing more heat evaporated to the system, and consequently higher temperature of the modified system compared to the PU-0. The maxT increases by about 20 °C with the addition of each POSS and appears at longer times compared to the PU-0  would react with isocyanate (–NCO) groups, therefore, the content of isocyanates which reacted with water and produced CO2 foaming gas would decrease, leading to the decreased foaming ratio and increased density. To sum up, the density of the composite foams increased with the incorporation of POSS filler in the PU system.Apparent density is one of the most important parameters to control the physical, mechanical and thermal properties of the RPUFs which has influence on their performance and applications. The values of density of prepared foams are presented in The cell morphology is one of the most important factors determining the physico-mechanical properties of RPUFs ,51. The As observed from the micrograph of the neat PU-0 a,b, the ImageJ software from SEM images and the median values are summarized in The values of the cell size of the RPUFs were statistically analyzed by means of The mechanical properties of RPUFs depend primarily on the cells’ morphology with the strength being higher in the direction of foam expansion. In −3 and compressive strengths of 309–351 kPa, which are well in the range exhibited by conventional commercial foams that present densities in the 15–130 kg m−3 range and compressive strength values in the range 200–220 kPa (for RPUFs at a density of 40 kg m−3) [The compression modulus and compressive strength of RPUFs are presented in  kg m−3) ,65. Basefσ) and apparent density is observed as well  of RPUFs in all cases. The reason is due to the presence of POSS aggregates within the PU matrix, which may act as defects during the tensile testing process and decrease fε of foam composites.As in the case of compression results presented in  as well . It can −2, respectively. This behavior is related to the good interface reinforcement matrix and the generation of fracture paths through the POSS-reinforced RPUFs. Thus, the deformability of the RPUFs matrix is reduced, which in turn affects the ductility in the foam surface. With this effect, the foam composite tends to form a more rigid structure and decrease the concentration of POSS, thus reducing the foam’s energy absorption, resulting in greater impact strength.The correlation between impact strength and apparent density is observed as well . With thgT, which corresponds to the maximum value of the curve loss tangent (tanδ) versus temperature. Compared to the RPUFs modified with TS-POSS and OH-POSS, RPUFs containing AP-POSS are characterized by higher gT. Wu et al. [gT of RPUFs reflects the rigidity of the polymer matrix which is a function of the isocyanate index, cross-link density and aromaticity level of the RPUFs. Given that the isocyanate index has been held constant in this study, the increase in the gT for POSS-modified samples must be a reflection of the increased aromaticity and cross-link density due to the presence of the POSS [tanδ peak is often assumed to be due to broader distribution in molecular weight between crosslinking points or heterogeneities in the network structures [The dynamic mechanical behavior of RPUFs as a function of the temperature is shown in u et al.  have shothe POSS . Moreoveructures .E’) as compared to PU-0. It can be concluded that the addition of all POSS has significantly increased the E’ of PU and consequently the stiffness of studied composites is also enhanced. This is due to the presence of filler in the PU matrix as well as higher viscosity of the modified systems, which imposes serious limits on the mobility of polymer chains, affecting their higher stiffness. Similar results are reported in the literature [In terature ,69.The thermal degradation of pure polyurethane foam and hybrid composites was monitored by TGA thermograms as displayed in It can be observed that the addition of fillers affects the thermal stability of RPUF . POSS usThe reduction of thermal stability can be attributed to non-homogeneous dispersion of POSS and changes in cross-link density . ConfirmFor RPUFs, often used as construction materials, dimensional stability, as well as affinity for water, is a very important parameter. Polyhedral oligomeric silsesquioxanes significantly affected the hydrophobicity of the foams . RegardiRPUFs were successfully reinforced using POSS with hydroxyl and amino groups. The impact of POSSs on thermal properties, dynamic mechanical properties, physico-mechanical properties , foaming parameters and morphology of RPUFs was examined. The presented results indicate that the addition of AP-POSS, TS-POSS, and OH-POSS in the range of 0.5 wt.% influences the morphology of analyzed foams and consequently their further mechanical and thermal properties. It was noticed that RPUFs modified with AP-POSS are characterized by smaller and more regular polyurethane cells. This suggests better compatibility between PU foam matrix and AP-POSS compared with other fillers. This results in significant improvement of physico-mechanical properties and thermal stability of composites with AP-POSS. For example, compared to the RPUFs modified with OH-POSS and TS-POSS, composition with 0.5 wt.% of the AP-POSS showed greater compressive strength (351 kPa) and higher flexural strength (0.469 MPa). However, the highest hydrophobicity showed OH-PU foams, which were characterized by the greatest contact angle (140°) and less water uptake (11.2% after 24 h).


This work reports the main conclusions of a study on the mechanical behavior of concrete under ISO 834 fire with different cooling methods. The aim of this research was to provide reliable data for the analysis of structures damaged by fire. The experimental program used cylindrical concrete test specimens subjected to ISO 834 heating in a furnace up to maximum gas temperatures of 400, 500, 600, 700, and 800 °C. The compressive strength was measured in three situations: (a) at the different temperature levels reached in the furnace; (b) after a natural cooling process; and (c) after aspersion with water at ambient temperature. The results indicate that the concrete residual compressive strength is fairly dependent on the maximum temperature reached in the furnace and revealed that concrete of a lower strength preserved relatively higher levels of strength. The cooling method significantly influenced the strength, albeit at a lower intensity. In all cases, the residual strength remained in the range of 38% to 67% of the strength at ambient temperature. The statistical analysis showed that the data obtained by the experimental program are significant and confirmed the influence of the conditions imposed on the residual strength. In recent decades, the study of concrete during and after a fire has been scant in Brazil. In the mid-1970s, after two buildings were damaged by fire, the development of fire safety technology became a challenge for engineers and fire authorities. A technical standard for the design of concrete structures under fire was published in 1976. It required fire resistance times for building structures, depending on their height and occupancy. This standard prescribed adequate concrete covering according to the required times for fire resistance. In general, considering low- and medium-rise buildings in Brazil (up to 15 floors), normal coverings of 25 mm were sufficient to achieve the desired fire resistance. In Europe and the United States, design guidelines are available in the Eurocodes (EN 1992-1-2) and American Standards (ACI/TMS 216.1-14). These standards emphasize the structural fire resistance of buildings and explicitly incorporate the verification of structural elements under fire conditions. A fire in a building can have huge consequences, putting human lives at risk. Nonetheless, concrete structures have a relatively high fire resistance and can often survive fire exposure. As such, it is relevant to investigate how to repair the damaged parts of a structure after a fire and thus avoid the costs of demolition and reconstruction. To estimate repair costs, it is necessary to assess the damage to a structure caused by fire [Several authors have reported studies on the influence of a fire on concrete properties, with the residual compressive strength being one of the most important. There is a great variety of reported research considering the influence of parameters such as the water cement ratio, aggregate density, microsilica rate, heating process, state of initial stress, and state of stress during tests on the residual strength of concrete ,3,4,5,6.Xotta et al. , Sinaie Therefore, the effect of high temperatures on the strength of normal concrete has been extensively investigated. Nevertheless, it is important to mention that the influence of the cooling process after high temperature exposure has been much less investigated, although the compressive strength is significantly influenced by the cooling method. Depending on the cooling method, an additional strength reduction up to 38% occurs for water-cooled specimens compared to slowly cooled specimens. However, the difference between cooling methods decreases when the concrete is exposed to higher temperatures. This is due to the bond strength between concrete and the reinforcement, which is very sensitive to high temperatures and subsequent cooling. A gradual decrease of the bond strength is observed until 600 °C . Bingöl In this context, it is essential to understand the behavior of concrete structures affected by fire and the influence of heat on building materials. Several studies have evaluated the residual properties of cement-based materials after a high temperature, whilst others have investigated the physical and chemical changes occurring at the microstructural level. However, comprehensive studies evaluating the influence of cooling methods on the mechanical properties of concrete are scarce in the literature. Therefore, the research reported here aims to broaden the knowledge on the subject, providing new results, and was designed to answer two questions: How is the residual strength of concrete under ISO 834 fires affected by the strength at ambient temperatures? To what extent does the cooling of hot concrete specimens affect the residual strength? These questions are particularly important for engineers and fire brigade officials when examining fire-damaged concrete structures.ck) of 15, 21, 25, and 35 MPa with water/cement ratios of 0.620, 0.517, 0.458, and 0.338, respectively. To determine the optimal ratio between sand and gravel, the modified Reilly [G characteristic of aggregates following the Reilly [1 = 4.2031 and M2 = 0.3281. All specimens were kept at ambient humidity for 90 days until testing.The coarse aggregates were limestone with a maximum diameter of 25 mm and fineness modulus of 7.05. A sand of fineness modulus 2.68 and a cement ASTM Type III  with a cd Reilly  method ad Reilly  was usede Reilly  method wThe experimental program included 252 cylindrical concrete specimens that were 100 mm in diameter and 200 mm in height. Initially, the materials were separated and weighed according to the predefined proportions. Then, the components were mechanically mixed in the following order: coarse aggregates, sand, half of the water content, Portland cement, and the remaining water. After obtaining a homogeneous mixture, the fresh concrete was poured into the cylindrical molds. Manual densification was performed to reduce voids and air bubbles, ensuring a uniform mass. After 24 h of molding, the stiffened specimens were removed from the molds and allowed to wet cure for 28 days, which was a sufficient time for cement hydration reactions and strength gain.gmax) of 400, 500, 600, 700, and 800 °C (48 specimens at each temperature). A typical time–temperature curve of the furnace is shown in The concrete test specimens were subjected to ISO 834 heating in a cylindrical furnace of a 770 mm diameter and 800 mm height, up to maximum gas temperatures . The control group was tested at room temperature for each theoretical strength.2). The null effect was identified by the value zero, and values close to or greater than 1 indicated a broad effect. The observed power represented the robustness of the tests in identifying the differences between the groups analysed. Values close to one indicated a high test power.The consistency of the data obtained was evaluated by statistical analysis. The significance level adopted was 5% (0.05). Therefore, the analyses had a confidence interval of 95%. Univariate analysis of variance (ANOVA) using the F-ratio was chosen as the analysis technique. The impact of the conditions imposed on the compressive strength was measured by the size of the effect, using the variances and the parameter eta-square . For heating up to 800 °C, the residual compressive strength remained in the range of 38% to 67% of the ambient temperature strength. Natural air cooling resulted in lower decreases in strength, while the greatest variations were obtained with water spray. The hot test resulted in an intermediate condition among the others. Similar results were obtained by Chan, Luo, and Sun [In the first analysis, the strength had a similar trend. There was a decrease in strength as the temperature increased, especially between room temperature and 400 °C. This result seems to be reasonable considering that 400 °C was reached in all experiments, which is the temperature which corresponds to major damage to the concrete microstructure, as observed by several authors ,11,12. B and Sun . They cop < 0.05), which enabled the variables to be correlated analytically. The categorical variable test method (M), temperature (T), and theoretical strength (fck) were significant (p < 0.05), which indicates a relevant effect on the compressive strength. The effect size was moderate for the test method and more intense for the temperature and strength variables. However, the values obtained indicate a significant influence of the conditions on the strength. The power observed was the maximum for all situations evaluated. The adopted model can explain 88% of the observed values (coefficient of determination Rsquare = 0.880). Therefore, the obtained data are statistically significant and adequately explain the proposed problem.If there is no material fragmentation during heating, chemical and physical phenomena, acting in a synergistic manner, promote a decrease in strength. Under the three conditions evaluated, the mechanisms of chemical and structural degradation mostly occurred during heating. Therefore, the portion of lost strength related to cooling methods is governed by physical phenomena . Abrupt (1)The combined conditions  had a significant influence on the residual strength. In general, the worst-case scenario was obtained considering high temperatures in the order of 800 °C, sudden cooling with water spray, and higher theoretical strengths. The best-case scenario was obtained with a 400 °C temperature, slow air cooling, and the lowest theoretical strengths;(2)Sudden cooling with water spraying was the most severe condition, resulting in the greatest decreases in strength. This result can be explained by the sudden dimensional variation of the particulate constituents and the cementitious matrix, which generates cracks and weakens the ITZ;(3)Concretes with a higher theoretical strength showed a lower residual strength. A higher strength implied a denser cementitious matrix, a lower porosity, and more consistent ITZ. However, the accommodation of stresses and strains was impaired, resulting in more extensive cracking and more severe degradation of the material;(4)The higher the temperature, the greater the losses of strength due to the degradation of concrete by physical and chemical phenomena. During heating, there was evaporation of free water, the release of chemically bound water, phase changes, and chemical changes;(5)Statistical analysis showed that the values obtained by the mechanical tests were significant and that the conditions evaluated  significantly influenced the residual strength;(6)In future studies, it is suggested that changes in the structure of the concrete cement matrix are studied using a scanning electron microscope (SEM) to aggregate information about changes in the matrix as a function of heating and cooling processes.This work presents a case study on the effect of the cooling method on the mechanical behavior of concrete after a fire according to ISO 843. Based on the results of the experimental study, the following conclusions can be formulated: