An undergraduate experimental research program, involving the screening of efflux pump inhibitors, to improve student engagement in biomedical science

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Medical microbiology is a crossover field requiring a broad range of interdisciplinary skills and education. However, a systematic experimental training system based on the integration of science and education is lacking. Here, we present an antimicrobial resistance-based pharmaceutical developing research (APDR) program that broadly covers key topics, such as microorganism isolation and identification, drug sensitivity detection, protein expression and homology modeling, and molecular docking and new drug screening, with the potential for publishable research outcomes. Significant improvements in follow-up course scores in genetic engineering (18.5%), microbiology (6.9%), and molecular biology (10.7%) were observed among participants. Furthermore, 44.1% of participants demonstrated scholarly productivity as exemplified by authored or co-authored publications. Therefore, a systematic APDR program, integrating biochemistry, microbiology, molecular biology, bioinformatics, and pharmacology skills, offers students a positive learning experience and provides a pedagogical tool that can be adapted to improve students’ ability to engage in biomedical research. APDR program systematic research antimicrobial resistance efflux pump inhibitors higher education Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Microbiology and medical microbiology are two basic courses that invariably feature in biology and medical undergraduate degree qualifications. Advancements in the field of life science have led to rapid developments in biotechnology, with increasing applications in industrial engineering, agriculture, and medicine [1, 2]. The rate of scientific and technological progress presents challenges for university faculties in keeping pace with the latest teaching [3]. Additionally, the rise of interdisciplinary subcategories available to undergraduates, such as biomedical science, biomedical engineering, medical laboratory science, and molecular pharmacology, has increased the requirement for an overall understanding of microbiology and medical microbiology [4]. Thus, courses that integrate microbiology, biochemistry, molecular biology, bioinformatics, and pharmacology to improve the theoretical knowledge and experimental skills of undergraduates are urgently needed. Antimicrobial resistance (AMR) has emerged as a major threat to public health [5] and economic development [6] during this century. In particular, the overuse of antibiotics in cases of bacterial coinfection [7] and secondary infection in COVID-19 patients [8] has contributed to the rise of AMR [9]. Active efflux is one mechanism that bacteria have developed or acquired that confers resistance against antibiotics [10]. Thus, developing efflux pumps inhibitors (EPIs) as new pharmaceuticals is a promising and valid strategy for fighting resistance to antibiotics [11]. Drug research targeting AMR is a multi-disciplinary integration task, including biochemistry, microbiology, molecular biology, and pharmacology [12] and is therefore an ideal subject area for the systematic learning of modern biomedical techniques for undergraduate education. Participating in science is essential for undergraduate students to develop scientific literacy and critical thinking skills [13]. Programs based on concepts, content, topics, and expected student outcomes, rather than traditional course delineations, has been developed recently [14]. The development of course-based undergraduate research experiences (CUREs) is becoming a popular strategy for instructors to provide students with meaningful experiences by actively engaging them in scientific investigation [13]. Participation in CUREs can help students gain research skills and improve their attitude toward science [15]. With the data revolution in the life sciences, bioinformatic methods have been increasingly applied in biological research at the graduate level and have percolated into undergraduate curriculums [16]. Bioinformatics programming exercises for students can provide a visible, interactive means of learning molecular biology concepts [17]. However, the lack of a systematic experimental training system has hindered the training of innovative talent in this area. Enriching and improving experimental teaching content is imperative to provide high-quality undergraduate education. In this study, we developed an undergraduate research laboratory training course that integrated biochemistry, microbiology, molecular biology, bioinformatics, and pharmacology, designated AMR-based pharmaceutical developing research (APDR), which focuses on identifying new EPIs from traditional Chinese medicine (TCM). By participating in the APDR curriculum, students can engage in authentic research projects, understand the principles of protein structure prediction, molecular docking, the binding of enzymes and their inhibitors, target validation, and other key areas of knowledge based on EPI identification. Materials and Methods 2.1 Student background Students voluntarily participating in the APDR curriculum were enrolled at Shandong First Medical University and were majoring in biomedical science. They were randomly divided into 10 research groups (Group 1 – Group 10). Instructors actively engaged students in authentic research through a two semester-long project that introduced them to synthetic biomedical science, including biochemistry, microbiology, molecular biology, bioinformatics, and pharmacology techniques and principles. The research had the potential to generate publishable scientific discoveries. 2.2 APDR program design The second-year APDR program builds on the mastery of subject precursor courses (chemistry and biochemistry that were taught in the first year), before the third-year professional core course (such as molecular biology, microbiology, or genetic engineering) is undertaken. Polymyxins are one of a few remaining available active agents against carbapenem-resistant bacteria and are increasingly being used as last-line therapeutic options against several multidrug-resistant bacteria [18, 19]. The increasing incidence of polymyxin resistance has been reported, with significant clinical implications [20]. The scientific goal of this APDR program is the development of EPIs as potential pharmaceuticals to combat polymyxin-resistant bacteria. To achieve this, students begin a two-year authentic research project by initially selecting a polymyxin-resistant bacterium, investigating its resistance genes and polymyxin efflux mechanism, then designing and conducting an experiment to screen for EPI compounds. The specific framework is shown in Fig. 1. Each module has one primary research goal. Students learn a variety of experimental techniques, while achieving the research goal of each module. 2.2 Program modules The experimental strategy of the APDR program is comprised of four modules: Module 1: Isolation and genome analysis of bacterial strains to identify a polymyxin-resistant isolate (~5 weeks). Module 2: Selecting an efflux pump gene from the genome and verifying its influence on polymyxin resistance (~5 weeks). Module 3: Analyzing the mechanism of polymyxin effluxion (~2 weeks). Module 4: Screening EPI compounds from the Traditional Chinese Medicine Active Compound Library (~4 weeks). 2.2.1 Module 1: Isolating polymyxin-resistant strains and genome analysis In this module, students collected aquaculture sewage samples. The samples were spread onto Luria–Bertani (LB)-agar plates (0.5% yeast extract, 1% tryptone, 1% sodium chloride, 2% agar) containing 8 mg/L polymyxins (Sigma Co., Shanghai, China) and then incubated at 28°C for 24 h. All colonies with different phenotypes on the plates were selected and subcultured three times on LB-agar with polymyxins to obtain single colonies. Then, 16S rDNA was amplified by PCR and sequenced to verify the purity of the colony. Antimicrobial susceptibility tests were performed to determine the minimum inhibitory concentrations (MICs) of polymyxin B and polymyxin E. Data were analyzed based on breakpoints defined by the Clinical and Laboratory Standards Institute [21]. The genome sequence of the isolated strain was determined and then annotated using either the Prokaryotic Genome Annotation Pipeline of NCBI or the PATRIC server [22]. 2.2.2 Module 2: Selecting an efflux pump gene that contributes to polymyxin resistance In this module, antibiotic resistance genes were analyzed using the Antibiotic Resistance Genes Database [23]. Students selected an efflux pump gene from the genome to test whether it affects polymyxin resistance. Then, molecular simulation analysis was conducted to predict the efflux of polymyxins and homologous model construction was undertaken using Swiss-Model or Discovery Studio 2.0 [24]. Molecular docking, using polymyxins as ligands, was performed according to the CDOCKER protocol of Discovery Studio 2.0 [24]. The chemical bonds between efflux pumps and polymyxins were demonstrated by 3D and 2D methods. 2.2.3 Module 3: Verifying polymyxin resistance by heterogeneous expression The genes of efflux pumps, which interact with polymyxins, were amplified by PCR using primers possessing a Xba I restriction site at the 5′-end and a Hin d III restriction site at the 3′-end, and genomic DNA as a template. The promoter of the β-lactamase gene was amplified by PCR using primers possessing a Hin d III site at the 5′-end and an Xba I site at the 3′-end, and the pMD18-T vector as a template. The above two gene fragments were then fused using T4 DNA ligase after Hin d III digestion. After Xba I digestion, the fused fragment was cloned into the pMD18-T vector and transformed into E. coli DH5α for heterogeneous expression. The MICs of the resulting transformant were tested. 2.2.4 Module 4: Screening the Traditional Chinese Medicine Active Compound Library for EPIs The Traditional Chinese Medicine Active Compound Library (MedChemExpress, China) was screened for potential EPIs of the selected efflux pump using the CDOCKER protocol of Discovery Studio 2.0 [25] in the presence of polymyxin B (PMB). Then, the MICs of polymyxins in the presence of EPIs were determined to evaluate the influence of screened EPIs on polymyxin resistance. Verapamil and compounds selected from the TCM Active Compound Library (designated EPI-X) were used as EPI positive controls at final concentrations of 8.0 mg/L, 0.1 mg/L, 8.0 mg/L, 8.0 mg/L, and 8.0 mg/L, respectively. The MICs of polymyxins were calculated by the broth microdilution procedure based on the breakpoints defined by the Clinical and Laboratory Standards Institute [21]. 2.3 Publishable research Through our APDR program, each student has an opportunity to participate in publishable research and has the option of being an author on a paper describing the research. Student authorship is based on the following “competitive teamwork” criteria: (1) order of authorship follows the order of completion of experiments with a credible result, (2) authorship depends on contribution to the work. 2.4. Learning effect assessment of student experiences To evaluate the learning effect of our APDR program, two approaches were employed. One involved evaluating the impact on other courses, including molecular biology, microbiology, and genetic engineering; the other involved a student survey in which students rated the efficacy of the class as well as the perceived impact of their participation. The survey consisted of six items, each of which could be answered on a 5-point Likert scale (“1 = Strongly Disagree”, “2 = Disagree”, “3 = Neutral”, “4 = Agree”, and “5 = Strongly Agree”). The items were designed to assess the students’ overall perception regarding the main objectives of the practical training program: “Increased interest in the study of biochemistry”; “Increased interest in the study of microbiology”; “Increased interest in the study of molecular biology”; “Increased interest in the study of bioinformatics”; “Increased interest in the study of pharmacology”; “Mastered the strategies of antibiotic-resistant bacteria isolation”; “Promoted understanding of protein structure and homology modeling”; “Acquired the principles and applications of molecular docking and drug screening”; “Understood the application of EPIs”; “Enjoyed designing their own research study”; and “Interested in graduate school or an MD/PhD”. GraphPad Prism (version 8.0 for Windows, GraphPad Software, San Diego, CA, USA) was used for statistical analysis. Results 3.1 Isolating and identifying polymyxin-resistant strains Polymyxin-resistant strain isolation aims to develop experimental skills in opportunistic pathogen culture, isolation, and purification. During this process, a single colony of the polymyxin-resistant strain is required and the instructor explains to students the merits and demerits of the plate streak method and the gradient dilution method. Students then decide which experimental scheme to adopt. Strain identification is conducted by PCR amplification and sequencing of 16S rDNA. The instructor explains to students the possibility of nonspecific amplification during PCR and students are asked to perform optimization. At the end of this phase, students in the 10 groups had successfully isolated and identified polymyxin-resistant strains (Table 1). The 10 isolated strains showed varying degrees of drug resistance (MICs ranged from 12–96 mg/L) (Fig. 2). The instructor evaluated the value of sequencing based on the degree of drug resistance, and seven of the isolated strains (MICs ≥ 24 mg/L) were selected for sequencing (Table S1). The six students in the three groups for which isolates were not selected for sequencing were incorporated into the other groups randomly. Table 1 Isolated polymyxin-resistant strains Group Strain number Species GenBank accession no. Characters GC Content (%) 1 M202 Pandoraea pnomenusa CP041237 Gram-negative 64.79 2 PL22-3A Roseomonas suffusca / Gram-negative / 3 PL22-11A Roseomonas cervicalis CP115851-CP115855 Gram-negative 72.03 4 PL22-22A Chryseobacterium sp. CP115858 Gram-negative 36.12 5 PL22-1A Chryseobacterium gambrini CP115857 Gram-negative 36.02 6 VAN22-5A Chryseobacterium camelliae CP115859 Gram-negative 35.37 7 PL23A Bacillus safensis CP132599 Gram- positive 41.72 8 P23-5 Stenotrophomonas / Gram- positive / 9 PL22-8A Brevundimonas vesicularis / Gram-negative / 10 V23-7 Psychrobacter sp. CP132595- CP132597 Gram-negative 43.69 3.2 Selecting an efflux pump gene associated with polymyxins by molecular docking After regrouping, the seven groups were guided in the analysis of drug resistance genes in the genome and the selection of an efflux pump gene to test throughout the semester to determine whether it mediates polymyxin resistance. For this process, the Antibiotic Resistance Genes Database was presented by the instructor. As shown in Table 2, seven sequenced strains were selected for further analysis, 233 antibiotic resistance genes were identified, and each group selected an efflux pump gene to use in subsequent experiments. Table 2 Sequenced strains and their corresponding antibiotic resistance genes Strains Number of resistant genes Number of efflux pump genes Location of selected efflux pump genes M202 43 18 MFS type transporter 4911571-4912923 PL22-11A 31 3 MdtABC-TolC like efflux pump 1554828- 1557975 bp PL22-22A 30 9 ErmAB-TolC like efflux pump 6173-10307bp PL22-1A 27 11 Bcr/CflA family transporter 821211-819934bp VAN22-5A 25 6 AcrAB-TolC like efflux pump 2850656- 2856382bp PL23A 38 3 EbrAB like efflux pump 1712971- 1712984bp V23-7 39 5 MacAB like efflux pump 728797- 728898 bp Working in groups of two or three students, students evaluated the possibility of the candidate pumps being involved in polymyxin efflux by molecular docking experiments. During the docking process, the size of the “receptor box” often affects the docking results [26]. Thus, the whole protein was set as the receptor box. The ErmAB-TolC-like efflux pump from Pandoraea pnomenusa PL22-22A, the MFS-type transporter from Chryseobacterium sp. M202, and the AcrAB-TolC-like transporter from Colletotrichum camelliae VAN22-5A (designated CprABC, FKQ53_RS21695, and AcrGH-TolC, respectively) showed polymyxin efflux capacity (Fig. 3), and this was confirmed by the instructor. The other efflux pumps did not demonstrate polymyxin efflux ability. Thus, three efflux systems were hypothesized to pump out polymyxins. A second regrouping was undertaken and the four groups without a docking frame were incorporated into the other three groups randomly. 3.3 Verifying polymyxin resistance by heterogeneous expression To confirm the polymyxin resistance mediated by the three efflux system described above, the encoding genes were expressed in E. coli strain DH5α. Students first designed PCR primers to amplify the efflux system encoding genes through homologous recombination. The instructor facilitates an in-depth discussion regarding the technique of PCR primer design, and the students design their own primers. To confirm successful PCR amplification, students perform agarose gel electrophoresis on a sample of the PCR product. If a band of the appropriate size is observed, the remaining PCR product is purified. Students use the purified DNA to transform competent E. coli cells and then test the MICs. As expected, all recombinant strains (expressing CprABC, FKQ53_RS21695, and AcrGH-TolC) showed polymyxin resistance (Fig. 4). 3.4 Screening EPIs from TCMs Traditional Chinese medicines (TCMs) are a huge reservoir of natural products, with a long history of practical application and therefore a great potential for drug development. The discovery of novel drug candidates from TCMs and their extracts has become a research hotspot [27]. Students were asked to screen EPIs that may inhibit the activity of CprABC, FKQ53_RS21695, and AcrGH-TolC from the Traditional Chinese Medicine Active Compound Library. In total, 220 compounds (11 compounds for each student on average) were analyzed by the molecular docking method, and then intra-group and inter-group discussions were carried out successively. The instructor then evaluated, selected, and explained the candidates recommended by the students. Two protein inhibitors, baicalin and sanguinarine (Fig. 5A), were selected for the CprABC and AcrGH-TolC efflux pumps, respectively. For the FKQ53_RS21695 efflux pump, no potential inhibitors were generated from the Traditional Chinese Medicine Active Compound Library, but students identified verapamil, a previously reported inhibitor, that may present inhibitory function (Fig. 5A). As shown in Fig. 4A, baicalin, a flavonoid compound, affected the allostery of CprB and/or obstructed the substrate conduit, and thus inhibited extracellular polymyxin transport. Verapamil exhibited inhibition via competition for polymyxin binding sites (Fig. 5B). Sanguinarine, a benzophenanthridine alkaloid, was found to bind within the phenylalanine-rich cage of AcrH to form a hydrophobic trap to prevent polymyxin transport (Fig. 5C). Students participating in this project were trained to dock compounds from the Traditional Chinese Medicine Active Compound Library with efflux pumps. Images of the results for different efflux pumps are presented. (A) Chemical formulas of EPIs. (B) Docking of baicalin and CprB; (C) Docking of verapamil and FKQ53_RS21695; (D) Docking of sanguinarine and AcrH. 3.5 Publishable research outcomes Through this APDR curriculum, students have an opportunity to participate in publishable research, and have the option of being an author on a paper describing the research. The independent writing of the manuscript by the research group is encouraged. Student authorship is based on the following criteria: (1) intellectual contribution to the work, (2) direct involvement in performing the research, and (3) participation in writing or editing the manuscript. Instructors are responsible for verifying experimental results and revising articles. To date, two papers describing efflux pumps and EPIs (baicalin for CprABC and verapamil for FKQ53_RS21695) identified by students have been published [28, 29], with seven and six students having participated in these two publications, respectively. A manuscript describing the efflux pump sanguinarine and corresponding EPI AcrH is currently in preparation and seven students are involved. 3.6 Assessment of students’ experiences To evaluate the effectiveness of the program, we analyzed the academic performance of students in three core disciplinary courses. A statistically significant improvement in the genetic engineering scores was observed, with the intervention group demonstrating a marked improvement from a baseline mean score of 75.23 (SD=±9.851) to 89.15 (SD=±9.322), representing an 18.5% increase (p<0.001), compared with non-participating controls maintaining stable performance levels. We also observed a 6.9% increase in microbiology scores [76.63 (SD=±8.027) to 81.90 (SD=±9.016)], and a 10.7% increase in molecular biology scores [77.34 (SD=±8.282) to 85.61 (SD=±7.881)] (Fig. 6A). To date, 14 participants (44.1%) in the program cohort have demonstrated scholarly productivity through authored or co-authored publications (Fig. 6B). To further measure the effectiveness of the program, we administered an anonymous questionnaire using a 5-point Likert scale. Overall, the results of the Likert survey were positive, with over 90% of students reporting increased interest in biomedical science and the acquisition of valuable information for their future studies. In addition, over 80% of students reported an interest in future research and post-graduate education (Fig. 6C). Together, these data suggest that the course was positively viewed by students as both a valuable educational experience and an asset to their continued professional development. Discussion Participating in science in meaningful ways is an essential approach to cultivate scientific talent with high-level theoretical knowledge and cutting-edge experimental techniques with relevance in the areas of science, technology, engineering, and mathematics [13]. Undergraduate research experiences provide several well-documented benefits for participating students. However, outcomes are greatly limited by the inherent inefficiency of the traditional one-on-one mentoring process, with insufficient faculty mentors to accommodate all interested students [30]. CUREs are an efficient means by which to provide all students with an original research project [31, 32]. The increasing interactions and integration between the fields of medical microbiology, biochemistry, bioinformatics, and pharmacology have led to the need for another level of teaching and learning [33]. An innovation of education in terms of curriculum to improve scientific literacy and skills is necessary to respond to the dramatic and rapid transformation of biological science in recent decades [30]. Here, we provide an APDR undergraduate research curriculum based on AMR that integrates biochemistry, microbiology, molecular biology, bioinformatics, and pharmacology courses to enhance students’ experimental skills and scientific thinking. Curriculum integration is a method designed to enhance the possibilities for personal and social development through organization of the curriculum around significant problems and issues, collaboratively identified by educators and students, transcending subject-area boundaries [34]. During construction, the undergraduate APDR curriculum focused on how separate subjects are rearranged in a curriculum proposal without losing their specific features. Scientific research is currently the academic area most affected by the integration of different disciplines [35]. Our research group has experience in the integration of science and education at Shandong First Medical University, and the valuable lessons learnt have been applied here to the field of modern medical microbiology research and education. Theory and experimental methods, such as DNA and protein separation, microorganism isolation and identification, drug sensitivity detection, PCR, plasmid construction and protein expression, protein homology modeling, molecular docking, and new drug screening and validation, which are all skills that must be understood and mastered for students of biochemistry, microbiology, medical microbiology, molecular biology, bioinformatics, and pharmacology, are all included in the APDR curriculum. As contemporary society is built on scientific knowledge, it is crucial that the next generation of scientists develop new levels of scientific literacy and inter-disciplinary critical thinking skills [36]. If developed at the undergraduate level, the skills to initiate research activities and work collaboratively between disciplines can then be applied at the postdoctoral level [37]. Furthermore, the APDR research curriculum is an extensible program that could be expanded to graduate students by adding modules, such as drug modification, amino acid virtual mutation, and molecular dynamics. There are some limitations to this pedagogical research. First, the sample size of students was limited and future studies with a larger population are needed. The program could also be extended to undergraduate students that major in biotechnology, public health, and medical laboratory technology. Second, the system for evaluating outcomes requires improvement. The impact on undergraduate grade point average (GPA), aptitude tests, postgraduate admission rate, and participants’ future career choices will be considered in subsequent studies. Moreover, the impact of the teachers’ area of expertise on outcomes will also be analyzed. In summary, we have established a viable and replicable research curriculum for medical microbiology education. This curriculum not only enhances students’ comprehension of medical microbiology but also holds significant promise for their future endeavors in the field of drug development. Declarations Acknowledgments We thank Jennifer Smith, PhD, from Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript. Authors’ contributions Gongli Zong and Peipei Zhang led the project, data analysis, and wrote the manuscript. Yongan Wang and Lin Wang performed the research as instructors. Jingxiang Pang and Jihong Pan analyzed the data. Jiafang Fu and Guangxiang Cao co-led the project and revised the manuscript. Funding This work was supported by the Shandong Province Undergraduate Teaching Reform Research Project – Key Project [Z2024215], the Educational Teaching Reform Research Project of Shandong First Medical University [grant number XM2024007], and the National College Students’ Innovation and Entrepreneurship Training Program [grant number 1905, 2024]. Ethics declaration This study was reviewed and approved by the Institutional Review Board of the Biomedical Sciences College, under approval number: 2022-03. Ethics approval and consent to participate Not applicable Consent for publication All authors approved the manuscript. Competing interests The authors declare that they have no competing interests. References Yan J, Chen J, Mao X, Li Q. Improvements in skills and knowledge after a comprehensive ELISA teaching course for biotechnology undergraduates. Biochem Mol Biol Educ. 2023;51(4):418–27. 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Cult Stud Sci Educ. 2023;18(1):21–39. Chen L, Wang J, Zhang X. Application of discipline integration in the practical teaching of endodontic–restorative sequential treatment. In.: Research Square; 2023. Avramovska O, Rokop ME. A low-cost cure for CUREs: An undergraduate microbiology course engaging students in authentic research using publicly available datasets. Biochem Mol Biol Educ 2023. Yoshioka-Kobayashi T, Shibayama S. Determinants of Ph.D. progression: student’s abilities and lab local environment. High Educ. 2022;86(3):693–718. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 26 May, 2025 Read the published version in BMC Medical Education → Version 1 posted Editorial decision: Revision requested 18 Apr, 2025 Reviews received at journal 18 Apr, 2025 Reviews received at journal 12 Apr, 2025 Reviewers agreed at journal 28 Mar, 2025 Reviewers agreed at journal 28 Mar, 2025 Reviewers agreed at journal 27 Mar, 2025 Reviewers agreed at journal 27 Mar, 2025 Reviewers agreed at journal 26 Mar, 2025 Reviewers agreed at journal 25 Mar, 2025 Reviewers invited by journal 25 Mar, 2025 Editor assigned by journal 24 Mar, 2025 Submission checks completed at journal 24 Mar, 2025 First submitted to journal 20 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6267603","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":435977343,"identity":"b1f1a4d6-10d9-43ec-b6ec-bee9260d84ec","order_by":0,"name":"Gongli Zong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYDADPmbmAww8NmC2AXFa2JjZEhh40kjSwsBjQJwW+fbewy+/VNyxa2Pn+fzhTcK2xAb25m0SDDV3cGph7DmXZi1z5llyGzPvNsk5CbcTG3iOlUkwHHuGUwuzRI6ZsWTb4WQ2oBZm3h9ALUARCcaGw7i9gNDC8/gzD8gW+Tf4tfBI5Bg//Nh22A6ohUEarEWCB78WCZ4zZswMZw4nAAPZDOQX4zaetGKLhGO4tci39xh//FFx2J6f//BjYIjdlu1nP7zxxoca3FpA3pHmYWBIbIBzQUQCPg3AQPv4g4HBHr+aUTAKRsEoGNEAAJLeUXXvzJ04AAAAAElFTkSuQmCC","orcid":"","institution":"Shandong First Medical University \u0026 Shandong Academy of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Gongli","middleName":"","lastName":"Zong","suffix":""},{"id":435977344,"identity":"1c007360-46b1-4419-b1fd-022b9d4be19f","order_by":1,"name":"Peipei Zhang","email":"","orcid":"","institution":"Shandong First Medical University \u0026 Shandong Academy of Medical 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Sciences","correspondingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Wang","suffix":""},{"id":435977348,"identity":"47f3c388-bc54-4d37-a4c1-e3590a7c92bc","order_by":5,"name":"Jihong Pan","email":"","orcid":"","institution":"Shandong First Medical University \u0026 Shandong Academy of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jihong","middleName":"","lastName":"Pan","suffix":""},{"id":435977349,"identity":"dd4044b3-106c-43be-85f6-b7437a1d94e3","order_by":6,"name":"Jingxiang Pang","email":"","orcid":"","institution":"Shandong First Medical University \u0026 Shandong Academy of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jingxiang","middleName":"","lastName":"Pang","suffix":""},{"id":435977350,"identity":"69171f1c-7d37-4316-b048-c995ef724bb8","order_by":7,"name":"Guangxiang Cao","email":"","orcid":"","institution":"Shandong First Medical University \u0026 Shandong Academy of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Guangxiang","middleName":"","lastName":"Cao","suffix":""},{"id":435977351,"identity":"36db4813-290d-4310-901e-86a22adb817d","order_by":8,"name":"Jiafang Fu","email":"","orcid":"","institution":"Shandong First Medical University \u0026 Shandong Academy of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jiafang","middleName":"","lastName":"Fu","suffix":""}],"badges":[],"createdAt":"2025-03-20 08:23:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6267603/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6267603/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12909-025-07340-2","type":"published","date":"2025-05-26T15:57:31+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79830647,"identity":"005ebe8e-d783-4c45-965c-4e93f4d8a8fb","added_by":"auto","created_at":"2025-04-03 10:24:46","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":218205,"visible":true,"origin":"","legend":"\u003cp\u003eFramework of the modular experimental design for the semester-long EPI identification project\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6267603/v1/5ad540ba2b0ba4b3ce475eef.jpeg"},{"id":79831331,"identity":"c481cd85-add7-4070-8027-e8ba0816edaf","added_by":"auto","created_at":"2025-04-03 10:32:46","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":101853,"visible":true,"origin":"","legend":"\u003cp\u003eMinimum inhibitory concentrations (MICs) of polymyxins for the different isolated strains\u003c/p\u003e\n\u003cp\u003e*: Resistant to polymyxin B and polymyxin E. #: Resistant to polymyxin B.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6267603/v1/d8f1aeeae383c44616e84bf3.jpeg"},{"id":79831330,"identity":"0cbef123-ffa1-4559-b1be-aca3dd44d903","added_by":"auto","created_at":"2025-04-03 10:32:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":473946,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular docking results of representative efflux pumps with polymyxins\u003c/p\u003e\n\u003cp\u003e(A–C) Homology model of CprABC from \u003cem\u003eP. pnomenusa \u003c/em\u003ePL22-22A, FKQ53_RS21695 from \u003cem\u003eChryseobacterium\u003c/em\u003esp. M202, and AcrGH-TolC from \u003cem\u003eC. camelliae\u003c/em\u003eVAN22-5A. (D–F) Molecular docking of CprB, FKQ53_RS21695, and AcrH with polymyxins. PMB and CST are shown as sticks; PMB- and CST-binding sites are shown by surfaces; PMB- and CST-binding amino acids are shown by lines.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6267603/v1/408aaa610081240ba5de54d2.png"},{"id":79830645,"identity":"5db91ac2-13bc-40f2-a3f6-a2b5ab248d51","added_by":"auto","created_at":"2025-04-03 10:24:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":116629,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative MIC testing results\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6267603/v1/6fe554dc1275aad2a592970d.png"},{"id":79831332,"identity":"349feea6-612d-4c24-932a-95725026f4fd","added_by":"auto","created_at":"2025-04-03 10:32:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":247917,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative EPI screening and docking results\u003c/p\u003e\n\u003cp\u003eStudents participating in this project were trained to dock compounds from the Traditional Chinese Medicine Active Compound Library with efflux pumps. Images of the results for different efflux pumps are presented. (A) Chemical formulas of EPIs. (B) Docking of baicalin and CprB; (C) Docking of verapamil and FKQ53_RS21695; (D) Docking of sanguinarine and AcrH.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6267603/v1/e595280b31d373c74e07917d.png"},{"id":79831948,"identity":"5a4d3e20-e75c-4016-bf85-5536775ff466","added_by":"auto","created_at":"2025-04-03 10:40:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":386639,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of the outcomes of the experimental training program\u003c/p\u003e\n\u003cp\u003e(A) Improvement in microbiology, genetic engineering, and molecular biology scores between participants (orange markers) and non-participants (green markers). (B) Scholarly productivity among participants. The years (2021–2023) represent the year in which students’ matriculate. (C) Outcomes of anonymous questionnaire survey of program participants.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6267603/v1/f429c5edf5368031eef92b8b.png"},{"id":83782882,"identity":"d77de633-cf81-4b01-a4f4-9432ce699f06","added_by":"auto","created_at":"2025-06-02 16:08:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2394451,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6267603/v1/3250655c-3a49-43e7-a50d-e7f3dbfec6ba.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"An undergraduate experimental research program, involving the screening of efflux pump inhibitors, to improve student engagement in biomedical science","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMicrobiology and medical microbiology are two basic courses that invariably feature in biology and medical undergraduate degree qualifications. Advancements in the field of life science have led to rapid developments in biotechnology, with increasing applications in industrial engineering, agriculture, and medicine [1, 2]. The rate of scientific and technological progress presents challenges for university faculties in keeping pace with the latest teaching [3]. Additionally, the rise of interdisciplinary subcategories available to undergraduates, such as biomedical science, biomedical engineering, medical laboratory science, and molecular pharmacology, has increased the requirement for an overall understanding of microbiology and medical microbiology [4]. Thus, courses that integrate microbiology, biochemistry, molecular biology, bioinformatics, and pharmacology to improve the theoretical knowledge and experimental skills of undergraduates are urgently needed.\u003c/p\u003e\n\u003cp\u003eAntimicrobial resistance (AMR) has emerged as a major threat to public health [5] and economic development [6] during this century. In particular, the overuse of antibiotics in cases of bacterial coinfection [7] and secondary infection in COVID-19 patients [8] has contributed to the rise of AMR [9]. Active efflux is one mechanism that bacteria have developed or acquired that confers resistance against antibiotics\u0026nbsp;[10]. Thus,\u0026nbsp;developing\u0026nbsp;efflux pumps inhibitors (EPIs)\u0026nbsp;as\u0026nbsp;new pharmaceuticals\u0026nbsp;is a promising and valid strategy for\u0026nbsp;fighting resistance to antibiotics\u0026nbsp;[11].\u0026nbsp;Drug research targeting AMR is a multi-disciplinary integration task, including biochemistry, microbiology, molecular biology, and pharmacology\u0026nbsp;[12]\u0026nbsp;and is therefore an ideal subject area for the systematic learning of\u0026nbsp;modern biomedical techniques for\u0026nbsp;undergraduate\u0026nbsp;education.\u003c/p\u003e\n\u003cp\u003eParticipating in science is essential for undergraduate students\u0026nbsp;to develop scientific literacy and critical thinking skills [13]. Programs based on concepts, content, topics, and expected student outcomes, rather than traditional course delineations, has been developed recently [14]. The development of course-based undergraduate research experiences (CUREs) is becoming a popular strategy for instructors to provide students with meaningful experiences by actively engaging them in scientific investigation [13]. Participation in CUREs can help students gain research skills and improve their attitude toward science [15].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWith the data revolution in the life sciences, bioinformatic methods have been increasingly applied in biological research at the graduate level and have percolated into undergraduate curriculums [16].\u0026nbsp;Bioinformatics programming exercises for students can provide a visible, interactive means of learning molecular biology concepts\u0026nbsp;[17]. However, the lack of a systematic experimental training system\u0026nbsp;has hindered the training of innovative talent in this area.\u0026nbsp;Enriching and improving experimental teaching content is imperative to provide high-quality undergraduate education.\u003c/p\u003e\n\u003cp\u003eIn this study, we developed an undergraduate research laboratory training course that integrated biochemistry, microbiology, molecular biology, bioinformatics, and pharmacology, designated AMR-based pharmaceutical developing research (APDR), which focuses on identifying new EPIs from traditional Chinese medicine (TCM). By participating in the APDR curriculum, students can engage in authentic research projects, understand the principles of protein structure prediction, molecular docking, the binding of enzymes and their inhibitors, target validation, and other key areas of knowledge based on EPI identification.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Student background\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudents voluntarily participating in the APDR curriculum were enrolled at Shandong First Medical University and were majoring in biomedical science. They were randomly divided into 10 research groups (Group 1 \u0026ndash; Group 10). Instructors actively engaged students in authentic research through a two semester-long project that introduced them to synthetic biomedical science, including biochemistry, microbiology, molecular biology, bioinformatics, and pharmacology techniques and principles. The research had the potential to generate publishable scientific discoveries.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 APDR\u003c/strong\u003e \u003cstrong\u003eprogram\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003edesign\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe second-year APDR program builds on the mastery of subject precursor courses (chemistry and biochemistry that were taught in the first year), before the third-year professional core course (such as molecular biology, microbiology, or genetic engineering) is undertaken. Polymyxins are one of a few remaining available active agents against carbapenem-resistant bacteria and are increasingly being used as last-line therapeutic options against several multidrug-resistant bacteria [18, 19]. The increasing incidence of polymyxin resistance has been reported, with significant clinical implications [20]. The scientific goal of this APDR program is the development of EPIs as potential pharmaceuticals to combat polymyxin-resistant bacteria. To achieve this, students begin a two-year authentic research project by initially selecting a polymyxin-resistant bacterium, investigating its resistance genes and polymyxin efflux mechanism, then designing and conducting an experiment to screen for EPI compounds. The specific framework is shown in Fig. 1.\u003c/p\u003e\n\u003cp\u003eEach module has one primary research goal. Students learn a variety of experimental techniques, while achieving the research goal of each module.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Program modules\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental strategy of the APDR program is comprised of four modules: Module 1: Isolation and genome analysis of bacterial strains to identify a polymyxin-resistant isolate (~5 weeks). Module 2: Selecting an efflux pump gene from the genome and verifying its influence on polymyxin resistance (~5 weeks). Module 3: Analyzing the mechanism of polymyxin effluxion (~2 weeks). Module 4: Screening EPI compounds from the Traditional Chinese Medicine Active Compound Library (~4 weeks).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.1 Module 1: Isolating polymyxin-resistant strains and genome analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this module, students collected aquaculture sewage samples. The samples were spread onto Luria\u0026ndash;Bertani (LB)-agar plates (0.5% yeast extract, 1% tryptone, 1% sodium chloride, 2% agar) containing 8 mg/L polymyxins (Sigma Co., Shanghai, China) and then incubated at 28\u0026deg;C for 24 h. All colonies with different phenotypes on the plates were selected and subcultured three times on LB-agar with polymyxins to obtain single colonies. Then, 16S rDNA was amplified by PCR and sequenced to verify the purity of the colony. Antimicrobial susceptibility tests were performed to determine the minimum inhibitory concentrations (MICs) of polymyxin B and polymyxin E. Data were analyzed based on breakpoints defined by the Clinical and Laboratory Standards Institute [21]. The genome sequence of the isolated strain was determined and then annotated using either the Prokaryotic Genome Annotation Pipeline of NCBI or the PATRIC server [22].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.2 Module 2: Selecting an efflux pump gene that contributes to polymyxin resistance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this module, antibiotic resistance genes were analyzed using the Antibiotic Resistance Genes Database [23]. Students selected an efflux pump gene from the genome to test whether it affects polymyxin resistance. Then, molecular simulation analysis was conducted to predict the efflux of polymyxins and homologous model construction was undertaken using Swiss-Model or Discovery Studio 2.0 [24]. Molecular docking, using polymyxins as ligands, was performed according to the CDOCKER protocol of Discovery Studio 2.0 [24]. The chemical bonds between efflux pumps and polymyxins were demonstrated by 3D and 2D methods.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.3 Module 3:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eVerifying polymyxin resistance by heterogeneous expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genes of efflux pumps, which interact with polymyxins, were amplified by PCR using primers possessing a \u003cem\u003eXba\u0026nbsp;\u003c/em\u003eI restriction site at the 5\u0026prime;-end and a \u003cem\u003eHin\u003c/em\u003ed\u003cem\u003e\u0026nbsp;\u003c/em\u003eIII restriction site at the 3\u0026prime;-end, and genomic DNA as a template. The promoter of the \u0026beta;-lactamase gene was amplified by PCR using primers possessing a \u003cem\u003eHin\u003c/em\u003ed\u003cem\u003e\u0026nbsp;\u003c/em\u003eIII site at the 5\u0026prime;-end and an \u003cem\u003eXba\u0026nbsp;\u003c/em\u003eI site at the 3\u0026prime;-end, and the pMD18-T vector as a template. The above two gene fragments were then fused using T4 DNA ligase after \u003cem\u003eHin\u003c/em\u003ed\u003cem\u003e\u0026nbsp;\u003c/em\u003eIII digestion. After \u003cem\u003eXba\u0026nbsp;\u003c/em\u003eI digestion, the fused fragment was cloned into the pMD18-T vector and transformed into \u003cem\u003eE. coli\u003c/em\u003e DH5\u0026alpha; for heterogeneous expression. The MICs of the resulting transformant were tested.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.4 Module 4: Screening the Traditional Chinese Medicine Active Compound Library for EPIs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Traditional Chinese Medicine Active Compound Library (MedChemExpress, China) was screened for potential EPIs of the\u0026nbsp;selected efflux pump\u0026nbsp;using the\u0026nbsp;CDOCKER protocol of Discovery Studio 2.0\u0026nbsp;[25]\u0026nbsp;in the presence of\u0026nbsp;polymyxin B (PMB). Then, the MICs of polymyxins in the presence of EPIs were determined to evaluate the influence of screened EPIs on polymyxin resistance.\u0026nbsp;Verapamil\u0026nbsp;and compounds selected from the\u0026nbsp;TCM Active Compound Library\u0026nbsp;(designated\u0026nbsp;EPI-X)\u0026nbsp;were used as EPI positive controls at final concentrations of 8.0\u0026nbsp;mg/L, 0.1\u0026nbsp;mg/L,\u0026nbsp;8.0\u0026nbsp;mg/L, 8.0\u0026nbsp;mg/L,\u0026nbsp;and 8.0\u0026nbsp;mg/L,\u0026nbsp;respectively. The MICs of polymyxins\u0026nbsp;were\u0026nbsp;calculated by the broth microdilution procedure\u0026nbsp;based on the breakpoints defined by the Clinical and Laboratory Standards Institute\u0026nbsp;[21].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Publishable research\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThrough our APDR program, each student has an opportunity to participate in publishable research and has the option of being an author on a paper describing the research. Student authorship is based on the following \u0026ldquo;competitive teamwork\u0026rdquo; criteria: (1) order of authorship follows the order of completion of experiments with a credible result, (2) authorship depends on contribution to the work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4. Learning effect assessment of student experiences\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the learning effect of our APDR program, two approaches were employed. One involved evaluating the impact on other courses, including molecular biology, microbiology, and genetic engineering; the other involved a student survey in which students rated the efficacy of the class as well as the perceived impact of their participation. The survey consisted of six items, each of which could be answered on a 5-point Likert scale (\u0026ldquo;1\u0026thinsp;=\u0026thinsp;Strongly Disagree\u0026rdquo;, \u0026ldquo;2\u0026thinsp;=\u0026thinsp;Disagree\u0026rdquo;, \u0026ldquo;3\u0026thinsp;=\u0026thinsp;Neutral\u0026rdquo;, \u0026ldquo;4\u0026thinsp;= Agree\u0026rdquo;, and \u0026ldquo;5\u0026thinsp;=\u0026thinsp;Strongly Agree\u0026rdquo;). The items were designed to assess the students\u0026rsquo; overall perception regarding the main objectives of the practical training program: \u0026ldquo;Increased interest in the study of biochemistry\u0026rdquo;; \u0026ldquo;Increased interest in the study of microbiology\u0026rdquo;; \u0026ldquo;Increased interest in the study of molecular biology\u0026rdquo;; \u0026ldquo;Increased interest in the study of bioinformatics\u0026rdquo;; \u0026ldquo;Increased interest in the study of pharmacology\u0026rdquo;; \u0026ldquo;Mastered the strategies of antibiotic-resistant bacteria isolation\u0026rdquo;; \u0026ldquo;Promoted understanding of protein structure and homology modeling\u0026rdquo;; \u0026ldquo;Acquired the principles and applications of molecular docking and drug screening\u0026rdquo;; \u0026ldquo;Understood the application of EPIs\u0026rdquo;; \u0026ldquo;Enjoyed designing their own research study\u0026rdquo;; and \u0026ldquo;Interested in graduate school or an MD/PhD\u0026rdquo;. GraphPad Prism (version 8.0 for Windows, GraphPad Software, San Diego, CA, USA) was used for statistical analysis.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e3.1\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eIsolating and identifying polymyxin-resistant strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePolymyxin-resistant strain isolation aims to develop experimental skills in opportunistic pathogen culture, isolation, and purification. During this process, a single colony of the polymyxin-resistant strain is required and the instructor explains to students the merits and demerits of the plate streak method and the gradient dilution method. Students then decide which experimental scheme to adopt. Strain identification is conducted by PCR amplification and sequencing of 16S rDNA. The instructor explains to students the possibility of nonspecific amplification during PCR and students are asked to perform optimization.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAt the end of this phase, students in the 10 groups had successfully isolated and identified polymyxin-resistant strains (Table 1). The 10 isolated strains showed varying degrees of drug resistance (MICs ranged from 12\u0026ndash;96 mg/L) (Fig. 2). The instructor evaluated the value of sequencing based on the degree of drug resistance, and seven of the isolated strains (MICs \u0026ge; 24 mg/L) were selected for sequencing (Table S1). The six students in the three groups for which isolates were not selected for sequencing were incorporated into the other groups randomly.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 1 Isolated polymyxin-resistant strains\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003eStrain number\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003eSpecies\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003eGenBank accession no.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eCharacters\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003eGC Content (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003eM202\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e\u003cem\u003ePandoraea pnomenusa \u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003eCP041237\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eGram-negative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e64.79\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003ePL22-3A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e\u003cem\u003eRoseomonas suffusca\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eGram-negative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003ePL22-11A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003eRoseomonas cervicalis \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003eCP115851-CP115855\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eGram-negative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e72.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003ePL22-22A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e\u003cem\u003eChryseobacterium sp.\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003eCP115858\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eGram-negative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e36.12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003ePL22-1A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e\u003cem\u003eChryseobacterium gambrini\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026nbsp;CP115857\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eGram-negative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e36.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003eVAN22-5A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e\u003cem\u003eChryseobacterium camelliae\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026nbsp;CP115859\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eGram-negative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e35.37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003ePL23A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus safensis\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003eCP132599\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eGram- positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e41.72\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003eP23-5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e\u003cem\u003eStenotrophomonas\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eGram- positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003ePL22-8A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e\u003cem\u003eBrevundimonas vesicularis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eGram-negative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003eV23-7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e\u003cem\u003ePsychrobacter\u0026nbsp;\u003c/em\u003esp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003eCP132595- CP132597\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eGram-negative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e43.69\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e3.2\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSelecting an efflux pump gene associated with polymyxins by molecular docking\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter regrouping, the seven groups were guided in the analysis of drug resistance genes in the genome and the selection of an efflux pump gene to test throughout the semester to determine whether it mediates polymyxin resistance. For this process, the Antibiotic Resistance Genes Database was presented by the instructor. As shown in Table 2, seven sequenced strains were selected for further analysis, 233 antibiotic resistance genes were identified, and each group selected an efflux pump gene to use in subsequent experiments.\u003c/p\u003e\n\u003cp\u003eTable 2 Sequenced strains and their corresponding antibiotic resistance genes\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eStrains\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003eNumber of resistant genes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eNumber of efflux pump genes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 202px;\"\u003e\n \u003cp\u003eLocation of selected efflux pump genes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eM202\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 202px;\"\u003e\n \u003cp\u003eMFS type transporter\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e4911571-4912923\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003ePL22-11A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 202px;\"\u003e\n \u003cp\u003eMdtABC-TolC like efflux pump\u003c/p\u003e\n \u003cp\u003e1554828-\u0026nbsp;1557975 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003ePL22-22A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 202px;\"\u003e\n \u003cp\u003eErmAB-TolC like efflux pump\u003c/p\u003e\n \u003cp\u003e6173-10307bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003ePL22-1A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 202px;\"\u003e\n \u003cp\u003eBcr/CflA family\u0026nbsp;transporter\u003c/p\u003e\n \u003cp\u003e821211-819934bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eVAN22-5A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 202px;\"\u003e\n \u003cp\u003eAcrAB-TolC like efflux pump\u003c/p\u003e\n \u003cp\u003e2850656-\u0026nbsp;2856382bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003ePL23A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 202px;\"\u003e\n \u003cp\u003eEbrAB like efflux pump\u003c/p\u003e\n \u003cp\u003e1712971-\u0026nbsp;1712984bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003eV23-7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 202px;\"\u003e\n \u003cp\u003eMacAB like efflux pump\u003c/p\u003e\n \u003cp\u003e728797-\u0026nbsp;728898 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eWorking in groups of two or three students, students evaluated the possibility of the candidate pumps being involved in polymyxin efflux by molecular docking experiments. During the docking process, the size of the \u0026ldquo;receptor box\u0026rdquo; often affects the docking results [26]. Thus, the whole protein was set as the receptor box. The ErmAB-TolC-like efflux pump from \u003cem\u003ePandoraea pnomenusa\u0026nbsp;\u003c/em\u003ePL22-22A, the MFS-type transporter from \u003cem\u003eChryseobacterium\u003c/em\u003e sp. M202, and the AcrAB-TolC-like transporter from \u003cem\u003eColletotrichum\u003c/em\u003e\u003cem\u003e\u0026nbsp;camelliae\u003c/em\u003e VAN22-5A (designated CprABC, FKQ53_RS21695, and AcrGH-TolC, respectively) showed polymyxin efflux capacity (Fig. 3), and this was confirmed by the instructor. The other efflux pumps did not demonstrate polymyxin efflux ability. Thus, three efflux systems were hypothesized to pump out polymyxins. A second regrouping was undertaken and the four groups without a docking frame were incorporated into the other three groups randomly.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eVerifying polymyxin resistance by heterogeneous expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo confirm the polymyxin resistance mediated by the three efflux system described above, the encoding genes were expressed in \u003cem\u003eE. coli\u003c/em\u003e strain DH5\u0026alpha;. Students first designed PCR primers to amplify the efflux system encoding genes through homologous recombination. The instructor facilitates an in-depth discussion regarding the technique of PCR primer design, and the students design their own primers. To confirm successful PCR amplification, students perform agarose gel electrophoresis on a sample of the PCR product. If a band of the appropriate size is observed, the remaining PCR product is purified. Students use the purified DNA to transform competent \u003cem\u003eE. coli\u003c/em\u003e cells and then test the MICs. As expected, all recombinant strains (expressing CprABC, FKQ53_RS21695, and AcrGH-TolC) showed polymyxin resistance (Fig. 4).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eScreening EPIs from TCMs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTraditional Chinese medicines (TCMs) are a huge reservoir of natural products, with a long history of practical application and therefore a great potential for drug development. The discovery of novel drug candidates from TCMs and their extracts has become a research hotspot [27]. Students were asked to screen EPIs that may inhibit the activity of CprABC, FKQ53_RS21695, and AcrGH-TolC from the Traditional Chinese Medicine Active Compound Library. In total, 220 compounds (11 compounds for each student on average) were analyzed by the molecular docking method, and then intra-group and inter-group discussions were carried out successively. The instructor then evaluated, selected, and explained the candidates recommended by the students. Two protein inhibitors, baicalin and sanguinarine (Fig. 5A), were selected for the CprABC and AcrGH-TolC efflux pumps, respectively. For the FKQ53_RS21695 efflux pump, no potential inhibitors were generated from the Traditional Chinese Medicine Active Compound Library, but students identified verapamil, a previously reported inhibitor, that may present inhibitory function (Fig. 5A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs shown in Fig. 4A, baicalin, a flavonoid compound, affected the allostery of CprB and/or obstructed the substrate conduit, and thus inhibited extracellular polymyxin transport. Verapamil exhibited inhibition via competition for polymyxin binding sites (Fig. 5B). Sanguinarine, a benzophenanthridine alkaloid, was found to bind within the phenylalanine-rich cage of AcrH to form a hydrophobic trap to prevent polymyxin transport (Fig. 5C).\u003c/p\u003e\n\u003cp\u003eStudents participating in this project were trained to dock compounds from the Traditional Chinese Medicine Active Compound Library with efflux pumps. Images of the results for different efflux pumps are presented. (A)\u0026nbsp;Chemical formulas of EPIs. (B) Docking of baicalin and CprB; (C) Docking of verapamil and FKQ53_RS21695; (D) Docking of sanguinarine and\u0026nbsp;AcrH.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Publishable research outcomes\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThrough this APDR curriculum, students have an opportunity to participate in publishable research, and have the option of being an author on a paper describing the research. The independent writing of the manuscript by the research group is encouraged. Student authorship is based on the following criteria: (1) intellectual contribution to the work, (2) direct involvement in performing the research, and (3) participation in writing or editing the manuscript. Instructors are responsible for verifying experimental results and revising articles. To date, two papers describing efflux pumps and EPIs (baicalin for CprABC and verapamil for FKQ53_RS21695) identified by students have been published [28, 29], with seven and six students having participated in these two publications, respectively. A manuscript describing the efflux pump sanguinarine and corresponding EPI AcrH is currently in preparation and seven students are involved.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 Assessment of students\u0026rsquo; experiences\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the effectiveness of the program, we analyzed the academic performance of students in three core disciplinary courses. A statistically significant improvement in the genetic engineering scores was observed, with the intervention group demonstrating a marked improvement from a baseline mean score of 75.23 (SD=\u0026plusmn;9.851) to 89.15 (SD=\u0026plusmn;9.322), representing an 18.5% increase (p\u0026lt;0.001), compared with non-participating controls maintaining stable performance levels. We also observed a 6.9% increase in microbiology scores [76.63 (SD=\u0026plusmn;8.027) to 81.90 (SD=\u0026plusmn;9.016)], and a 10.7% increase in molecular biology scores [77.34 (SD=\u0026plusmn;8.282) to 85.61 (SD=\u0026plusmn;7.881)] (Fig. 6A). To date, 14 participants (44.1%) in the program cohort have demonstrated scholarly productivity through authored or co-authored publications (Fig. 6B).\u003c/p\u003e\n\u003cp\u003eTo further measure the effectiveness of the program, we administered an anonymous questionnaire using a 5-point Likert scale. Overall, the results of the Likert survey were positive, with over 90% of students reporting increased interest in biomedical science and the acquisition of valuable information for their future studies. In addition, over 80% of students reported an interest in future research and post-graduate education (Fig. 6C). Together, these data suggest that the course was positively viewed by students as both a valuable educational experience and an asset to their continued professional development.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eParticipating in science in meaningful ways is an essential approach to cultivate scientific talent with high-level theoretical knowledge and cutting-edge experimental techniques with relevance in the areas of science, technology, engineering, and mathematics [13]. Undergraduate research experiences provide several well-documented benefits for participating students. However, outcomes are greatly limited by the inherent inefficiency of the traditional one-on-one mentoring process, with insufficient faculty mentors to accommodate all interested students [30]. CUREs are an efficient means by which to provide all students with an original research project [31, 32].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe increasing interactions and integration between the fields of medical microbiology, biochemistry, bioinformatics, and pharmacology have led to the need for another level of teaching and learning [33]. An innovation of education in terms of curriculum to\u0026nbsp;improve scientific literacy and skills\u0026nbsp;is\u0026nbsp;necessary to respond to the dramatic and rapid transformation of biological science in recent decades\u0026nbsp;[30]. Here,\u0026nbsp;we provide an APDR\u0026nbsp;undergraduate research\u0026nbsp;curriculum based on AMR\u0026nbsp;that integrates\u0026nbsp;biochemistry, microbiology, molecular biology, bioinformatics, and pharmacology\u0026nbsp;courses\u0026nbsp;to\u0026nbsp;enhance students’ experimental skills and scientific thinking.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCurriculum integration is a method designed to enhance the possibilities for personal and social development through organization of the curriculum around significant problems and issues, collaboratively identified by educators and students, transcending subject-area boundaries [34]. During construction, the\u0026nbsp;undergraduate\u0026nbsp;APDR\u0026nbsp;curriculum focused on how separate subjects are rearranged in a curriculum proposal without losing their specific features. Scientific research is currently the academic area most affected by the integration of different disciplines\u0026nbsp;[35]. Our research group has experience in the integration of science and education at Shandong First Medical University, and the valuable lessons learnt have been applied here to the field of modern medical microbiology research and education. Theory and experimental methods, such as DNA and protein separation, microorganism isolation and identification, drug sensitivity detection, PCR, plasmid construction and protein expression, protein homology modeling, molecular docking, and new drug screening and validation, which are all skills that must be understood and mastered for students of biochemistry, microbiology, medical microbiology, molecular biology, bioinformatics, and pharmacology, are all included in the\u0026nbsp;APDR\u0026nbsp;curriculum.\u003c/p\u003e\n\u003cp\u003eAs contemporary society is built on scientific knowledge, it is crucial that the next generation of scientists develop new levels of scientific literacy and inter-disciplinary critical thinking skills [36]. If developed at the undergraduate level, the skills to initiate research activities and work collaboratively between disciplines can then be applied at the postdoctoral level [37].\u0026nbsp;Furthermore, the\u0026nbsp;APDR\u0026nbsp;research\u0026nbsp;curriculum is an extensible program that could be\u0026nbsp;expanded to\u0026nbsp;graduate students by adding modules, such as drug modification, amino acid virtual mutation, and molecular dynamics.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThere are some limitations to this pedagogical research. First,\u0026nbsp;the sample size of students was limited and future studies with a larger population are needed. The program could also be extended to undergraduate students that major in biotechnology, public health,\u0026nbsp;and\u0026nbsp;medical laboratory technology. Second, the system for evaluating outcomes requires improvement. The impact on undergraduate\u0026nbsp;grade point average (GPA), aptitude tests, postgraduate admission rate, and participants’ future career choices will be considered in subsequent studies. Moreover, the impact of the teachers’ area of expertise on outcomes will also be analyzed.\u003c/p\u003e\n\u003cp\u003eIn summary, we have established a viable and replicable research\u0026nbsp;curriculum\u0026nbsp;for medical microbiology education. This\u0026nbsp;curriculum\u0026nbsp;not only enhances students’ comprehension of medical microbiology but also holds significant promise for their future endeavors in the field of drug development.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Jennifer Smith, PhD, from Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGongli Zong and Peipei Zhang led the project, data analysis, and wrote the manuscript. Yongan Wang and Lin Wang performed the research as instructors. Jingxiang Pang and Jihong Pan analyzed the data. Jiafang Fu and Guangxiang Cao co-led the project and revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Shandong Province Undergraduate Teaching Reform Research Project \u0026ndash; Key Project [Z2024215], the Educational Teaching Reform Research Project\u0026nbsp;of Shandong First Medical University\u0026nbsp;[grant number XM2024007],\u0026nbsp;and\u0026nbsp;the\u0026nbsp;National College Students\u0026rsquo; Innovation and Entrepreneurship Training Program\u0026nbsp;[grant number\u0026nbsp;1905,\u0026nbsp;2024].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was reviewed and approved by the Institutional Review Board of the Biomedical Sciences College, under approval number: 2022-03.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYan J, Chen J, Mao X, Li Q. 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Freire's hope in radically changing times: a dialogue for curriculum integration from science education to face the climate crisis. Cult Stud Sci Educ. 2023;18(1):21\u0026ndash;39.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen L, Wang J, Zhang X. Application of discipline integration in the practical teaching of endodontic\u0026ndash;restorative sequential treatment. In.: Research Square; 2023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAvramovska O, Rokop ME. A low-cost cure for CUREs: An undergraduate microbiology course engaging students in authentic research using publicly available datasets. Biochem Mol Biol Educ 2023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoshioka-Kobayashi T, Shibayama S. Determinants of Ph.D. progression: student\u0026rsquo;s abilities and lab local environment. High Educ. 2022;86(3):693\u0026ndash;718.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-medical-education","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"meed","sideBox":"Learn more about [BMC Medical Education](http://bmcmededuc.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/meed/default.aspx","title":"BMC Medical Education","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"APDR program, systematic research, antimicrobial resistance, efflux pump inhibitors, higher education","lastPublishedDoi":"10.21203/rs.3.rs-6267603/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6267603/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eParticipating in scientific research is essential for undergraduate students that major in biotechnology, public health, biomedical science, and medical laboratory technology. Medical microbiology is a crossover field requiring a broad range of interdisciplinary skills and education. However, a systematic experimental training system based on the integration of science and education is lacking. Here, we present an antimicrobial resistance-based pharmaceutical developing research (APDR) program that broadly covers key topics, such as microorganism isolation and identification, drug sensitivity detection, protein expression and homology modeling, and molecular docking and new drug screening, with the potential for publishable research outcomes. Significant improvements in follow-up course scores in genetic engineering (18.5%), microbiology (6.9%), and molecular biology (10.7%) were observed among participants. Furthermore, 44.1% of participants demonstrated scholarly productivity as exemplified by authored or co-authored publications. Therefore, a systematic APDR program, integrating biochemistry, microbiology, molecular biology, bioinformatics, and pharmacology skills, offers students a positive learning experience and provides a pedagogical tool that can be adapted to improve students\u0026rsquo; ability to engage in biomedical research.\u003c/p\u003e","manuscriptTitle":"An undergraduate experimental research program, involving the screening of efflux pump inhibitors, to improve student engagement in biomedical science","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-03 10:24:41","doi":"10.21203/rs.3.rs-6267603/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-18T14:39:18+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-18T11:29:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-12T18:06:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"338809312596743447403142698430629510864","date":"2025-03-28T14:13:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"305259664898579196713732385407021864698","date":"2025-03-28T14:11:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"338614647953274630333055484992916366597","date":"2025-03-27T21:47:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"53838653842390607155130823613190470631","date":"2025-03-27T21:21:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"269375632936733703580265781635493265094","date":"2025-03-26T16:18:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"161190813146670971636832372501997117325","date":"2025-03-25T22:20:04+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-25T21:20:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-24T14:00:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-24T13:56:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Medical Education","date":"2025-03-20T08:10:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-medical-education","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"meed","sideBox":"Learn more about [BMC Medical Education](http://bmcmededuc.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/meed/default.aspx","title":"BMC Medical Education","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"05f707be-b448-47ed-bed2-1fa97aca239a","owner":[],"postedDate":"April 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-02T16:01:33+00:00","versionOfRecord":{"articleIdentity":"rs-6267603","link":"https://doi.org/10.1186/s12909-025-07340-2","journal":{"identity":"bmc-medical-education","isVorOnly":false,"title":"BMC Medical Education"},"publishedOn":"2025-05-26 15:57:31","publishedOnDateReadable":"May 26th, 2025"},"versionCreatedAt":"2025-04-03 10:24:41","video":"","vorDoi":"10.1186/s12909-025-07340-2","vorDoiUrl":"https://doi.org/10.1186/s12909-025-07340-2","workflowStages":[]},"version":"v1","identity":"rs-6267603","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6267603","identity":"rs-6267603","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}