Study on the Antibacterial Efficacy of Ceftazidime-Avibactam Combined with Aztreonam against Multidrug-Resistant Bacteria in Hypothermic Organ Preservation Solution

Study on the Antibacterial Efficacy of Ceftazidime-Avibactam Combined with Aztreonam against Multidrug-Resistant Bacteria in Hypothermic Organ Preservation Solution | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Study on the Antibacterial Efficacy of Ceftazidime-Avibactam Combined with Aztreonam against Multidrug-Resistant Bacteria in Hypothermic Organ Preservation Solution Yazhe Duan, Daqian Tang, Yuhong Li, Pei Zhang, Yuxiang Wan, Kang Wu, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7197658/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The use of antimicrobial agents in the preservation solution (PS) could prevent the bacterial transmission to recipients in organ transplantation. However, antibiotics may exhibit different decontamination efficacy in the hypothermic PS. This study aimed to evaluate the antibacterial activity of ceftazidime-avibactam (CAZ-AVI) combined with aztreonam (ATM) against multidrug-resistant bacteria (MDRB) at varying ratios and concentrations during 3-hour, 0–4℃ PS decontamination. Results For CRKP and CREC, the 2:1 ratio at 0.5× concentration (250 mg/L CAZ-AVI + 0.5 g/L ATM) showed significant antibacterial effects ( P = 0.002 and P < 0.001, respectively). For CRPA, efficacy was observed at the 1× concentration with a 2:1 ratio (500 mg/L CAZ-AVI + 1.0 g/L ATM; P = 0.022), while for CRAB, the 1:1 ratio at 0.5× concentration (125 mg/L CAZ-AVI + 0.5 g/L ATM; P < 0.001) was effective. The combination was only effective against MRSA at high concentrations (1000 mg/L CAZ-AVI + 2.0 g/L ATM; P < 0.001). Conclusions The combination of CAZ-AVI and ATM effectively decontaminates MDR Gram-negative bacteria in PS. The 2:1 ratio at baseline concentration is recommended for clinical use, with potential escalation of CAZ-AVI concentration if needed. Organ transplantation Multidrug-resistant bacteria Ceftazidime-avibactam Aztreonam Organ preservation solution Figures Figure 1 Figure 2 Figure 3 Introduction Organ transplantation is a critical treatment for end-stage organ failure, with numerous procedures performed annually to save patients’ lives. However, allografts may be contaminated by bacteria originating from donors or during the acquisition, preservation or transportation processes[ 1 – 3 ]. To prevent bacterial transmission to recipients, the addition of antimicrobial agents to preservation solution (PS) for decontamination has been adopted by transplant centers worldwide[ 1 , 4 , 5 ]. Nevertheless, multidrug-resistant bacteria (MDRB) pose significant challenges and the MDRB related graft contamination may severely compromises transplant outcomes[ 6 – 8 ]. Therefore, developing novel antimicrobial strategies to enhance decontamination efficiency against MDRB is imperative. Avibactam (AVI), a novel β-lactamase inhibitor, is effective against class A, C, and D β-lactamases (e.g., KPC, AmpC, and OXA-48) but exhibits no activity against class B metallo-β-lactamases (such as NDM and VIM) [9] . Aztreonam (ATM), a monobactam antibiotic, demonstrates intrinsic stability against NDM [10] and is easily hydrolyzed by extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases [11, 12] . The combination of these agents (CAZ-AVI/ATM) could enhance the antimicrobial effect against MDRB due to their complementary mechanisms[ 13 – 16 ]. The primary objective of this study is to investigate the bactericidal efficacy of this combination against MDRB in low-temperature PS, which remains undocumented. We will investigate whether the synergistic application of drugs at varying ratios and concentrations significantly reduces bacterial contamination in PS and determine the optimal drug combination for effective decontamination. Methods Materials CAZ-AVI for injection (250 mg; CAZ 200 mg + AVI 50 mg) was obtained from Qilu Pharmaceutical Co., Ltd. (Jinan, China). ATM for injection (1 g) was sourced from Luoxin Pharmaceutical Co., Ltd. (Linyi, China). The organ preservation solution, hypertonic citrate adenine solution II (HC-A II), was acquired from Shanghai Haini Pharmaceutical Co., Ltd. (Shanghai, China). Additional materials included: a DensiCHEK-plus densitometer (BioMérieux, France), Columbia blood agar plates (Φ90 mm; Kemajia Shanghai Microbe Technology Co., Ltd., Shanghai, China), disposable plastic spreaders (BS-PS-A; Labgic Technology Co., Ltd., Beijing, China), and sterile 96-well cell plates (Corning Life Sciences Co., Ltd., Suzhou, China). Bacterial Strains and Culture Conditions Five clinically representative MDRB were selected for this study: carbapenem-resistant Klebsiella pneumoniae (CRKP), carbapenem-resistant Pseudomonas aeruginosa (CRPA), carbapenem-resistant Escherichia coli (CREC), carbapenem-resistant Acinetobacter baumannii (CRAB), and methicillin-resistant Staphylococcus aureus (MRSA). Clinical isolates were obtained from inpatients at Shanghai Changhai Hospital. All bacterial strains were isolated and cultured in the microbiology laboratory following the National Clinical Laboratory Practice Guidelines (4th edition). Species identification was performed using the MALDI Biotyper system (Bruker Daltonik, Germany). Antibiotic susceptibility testing was conducted via the disk diffusion method. After the bacteria were isolated and cultured from specimens, individual bacterial colonies were inoculated into the designated broth media, all samples underwent genetic testing by fluorescent PCR to determine bacterial genotypes/phenotypes and detect antibiotic resistance genes (Table 1 ). Prior to experiments, bacterial stocks were preserved at − 80°C. For reactivation, one frozen strain was subcultured twice on Columbia blood agar plates. A single colony was then aseptically suspended in PS, and the turbidity was adjusted to 0.5 McFarland standard using a densitometer. Table 1 Results of antibiotic resistance genes detection for various strains Strains Resistance Gene 1 Resistance Gene 2 Resistance Gene 3 CRKP CTX-M KPC NDM CRPA - - - CREC CTX-M NDM - CRAB OXA-23 - - MRSA - - - CRKP carbapenem-resistant Klebsiella pneumoniae , CRPA carbapenem-resistant Pseudomonas aeruginosa , CREC carbapenem-resistant Escherichia coli , CRAB carbapenem-resistant Acinetobacter baumannii , MRSA methicillin-resistant Staphylococcus aureus , CTX-M Cefotaxime-hydrolyzing β-lactamase-Munich, KPC Klebsiella pneumoniae carbapenemase, NDM New Delhi metallo-β-lactamase, OXA-23 Oxacillin-hydrolyzing β-lactamase-23 2.3. Definition of CAZ-AVI/ATM Ratios and Concentrations The baseline concentration was defined as 250 mg/L CAZ-AVI + 1 g/L ATM, and this ratio was considered as 1:1. Four drug ratios were established: 1:1, 1:2, 1:4, and 2:1. For each ratio, four concentration gradients were tested: 0.5×, 1×, 2×, and 4× the baseline concentration. Aliquots (50 µl) of each antibiotic solution were dispensed into 96-well plates and pre-chilled at 0–4°C for 24 hours prior to experimentation. The complete matrix of CAZ-AVI/ATM ratios and concentrations is detailed in Table 2 . Table 2 The ratios and concentrations of CAZ-AVI/ATM CAZ-AVI : ATM 0.5× 1× 2× 4× 1 : 1 125mg/L + 0.5g/L 250mg/L + 1.0g/L 500mg/L + 2.0g/L 1000mg/L + 4.0g/L 1 : 2 125mg/L + 1.0g/L 250mg/L + 2.0g/L 500mg/L + 4.0g/L 1000mg/L + 8.0g/L 1 : 4 125mg/L + 2.0g/L 250mg/L + 4.0g/L 500mg/L + 8.0g/L 1000mg/L + 16.0g/L 2 : 1 250mg/L + 0.5g/L 500mg/L + 1.0g/L 1000mg/L + 2.0g/L 2000mg/L + 4.0g/L Evaluation of MDRB Decontamination by CAZ-AVI/ATM Under 3-Hour Cold Storage Conditions Blank controls (antibiotic-free) and treatment groups were established with 6–9 replicates per condition. Bacterial suspensions were serially diluted with PS, and 50 µl aliquots were inoculated into pre-chilled 96-well plates. After thorough mixing, plates were maintained at 0–4°C for 3 hours to simulate clinical graft preservation conditions, yielding a final bacterial concentration of 1.5 × 10⁴ CFU/ml. After decontamination,10 µl aliquots from each well were plated onto Columbia blood agar plates, uniformly spread using sterile plastic spreaders (BS-PS-A), and incubated at 37°C for 20–24 hours. Colony-forming units (CFU) were quantified, and bacterial identity was confirmed by MALDI-TOF mass spectrometry. The optimal antibiotic ratio and minimum effective concentration were determined through statistical comparison of CFU reduction between treatment groups and blank controls. Statistical Analysis Statistical analyses were performed using IBM SPSS Statistics 24 and GraphPad Prism 9.5. Continuous data are presented as mean ± SD. Based on normality (Shapiro-Wilk test) and homogeneity of variance (Levene's test), we applied: One-way ANOVA with LSD test (normal distribution, equal variance), Welch ANOVA with Games-Howell test (normal distribution, unequal variance), Kruskal-Wallis test with Dunn's correction (non-normal distribution). A two-tailed P < 0.05 was considered significant. Results Ratio Selection of CAZ-AVI/ATM The antimicrobial efficacy of CAZ-AVI/ATM against CRKP was illustrated in Fig. 1. All tested ratios exhibited concentration-dependent bactericidal activity, with the 1:1 ratio demonstrating statistically significant reductions in colony counts compared to blank controls at concentrations ≥ 1× ( P = 0.016 for 1×, P = 0.009 for 2×, and P = 0.002 for 4×). The 1:2 and 1:4 ratios required higher concentrations (2× and 1×, respectively) to achieve statistical significance ( P = 0.022 and P = 0.039, respectively). Notably, the 2:1 ratio showed a better synergistic effect, exhibiting significant bacterial reduction even at the 0.5× concentration ( P = 0.002). These findings suggest that increasing the proportion of CAZ-AVI enhances synergistic antimicrobial activity more effectively than increasing ATM. Based on these results, the subsequent experiments focused on the 2:1, 1:1 and 1:4 groups to further investigate this synergistic pattern. Antibacterial Efficacy of CAZ-AVI/ ATM Against MDR-GNB Under 3-Hour Hypothermic Conditions For CRPA (Fig. 2A), both the 1:1 and 1:4 groups required ≥ 2× concentrations to demonstrate statistically significant differences compared to the blank control ( P = 0.009 and P = 0.001, respectively), while the 2:1 group showed significant differences at the 1× concentration ( P = 0.022). For CREC (Fig. 2B), the 1:4 and 2:1 groups exhibited significant bactericidal activity at the 0.5× concentration ( P = 0.017 and P < 0.001, respectively), the 1:1 group required ≥ 1× concentrations to achieve comparable effects ( P < 0.001). For CRAB (Fig. 2C), both the 1:1 and 2:1 groups showed significant CFU reduction versus blank controls at the lowest concentration (0.5×; P < 0.001 and P = 0.034, respectively), whereas the 1:4 group failed to demonstrate statistical significance at either 0.5× or 1× ( P = 0.356 and P = 0.117, respectively). Antibacterial Efficacy of CAZ-AVI/ATM Against MRSA Under 3-Hour Hypothermic Conditions As shown in Fig. 3, no statistically significant differences were observed among groups when using 1:1, 1:2, or 1:4 ratios ( P = 0.181, 0.697, and 0.112, respectively). However, when the ratio was adjusted to 2:1 and the concentration increased to ≥ 2×, statistically significant differences were achieved compared to the blank controls (both P < 0.001). Discussion The antimicrobial efficacy evaluation under hypothermic conditions presents unique pharmacological challenges. Existing evidence demonstrates that minimal bactericidal concentration (MBC) at 4°C may be hundreds to thousands of times higher than standard MBC values[ 17 ], underscoring the critical need for condition-specific antimicrobial assessments. This study evaluated the CAZ-AVI/ATM efficacy against five clinically significant MDRB strains during a 3-hour low-temperature (0–4°C) decontamination protocol using HC-A II PS. The findings established both the optimal drug ratio and the minimal effective concentration required for reducing bacterial contamination under clinically relevant preservation conditions. The preliminary investigation of CAZ-AVI/ATM against CRKP (Fig. 1) revealed that 1:1 or ATM-dominant ratios (1:2, and 1:4) achieved significant bacterial growth inhibition at 1× or 2× concentrations, whereas the CAZ-AVI-dominant ratio (2:1) demonstrated satisfactory bactericidal effects at 0.5× concentration, suggesting enhanced synergy with higher CAZ-AVI proportions. In subsequent experiments comparing the bactericidal efficacy of three ratios (1:1, 1:4, and 2:1) against CRPA, CREC, and CRAB, the results demonstrated that the CAZ-AVI-dominant ratio achieved effective inhibition at only 0.5× or 1× concentration while the 1:1 and ATM-dominant ratio required ≥ 2× concentration to inhibit bacterial growth. Specially, the ATM-dominant ratio exhibited inferior bactericidal efficacy against CRAB compared to the 1:1 at the same concentration. We further investigated the bactericidal efficacy against MRSA. The results were not satisfactory and only the CAZ-AVI-dominant ratio (2:1) exhibited bactericidal effects at higher concentrations (2× and 4×) (Fig. 3). These comprehensive results demonstrate that CAZ-AVI/ATM effectively inhibit these MDR GNB in PS under the condition of clinic decontamination procedure, with synergy enhanced by increasing CAZ-AVI ratio but limited enhancement by higher ATM ratio. Furthermore, CAZ-AVI has limited activity against Gram-positive organisms; however, sufficiently high concentration may achieve antibacterial effects. Considering the cost-effectiveness and antimicrobial efficacy, our findings identify the 2:1 ratio as optimal (compared with 1:1, 1:2, and 1:4), though minimum effective concentrations vary slightly among pathogens as detailed in Table 3 . The PS decontamination regimen does not rely only on antibiotics, and soaking and perfusion can also help remove pathogens. Recent evidence suggests that machine perfusion could enhance bacterial clearance through its closed-loop system, which maintains higher antibiotic concentrations within the organ while providing mechanical flushing[ 18 – 20 ]. Based on this study, we recommend adding 500mg CAZ-AVI and 1g ATM per liter of PS, with supplemental MRSA-targeted antibiotics if the donor was at high risk. Combining this protocol with machine perfusion may further improve bacterial clearance in contaminated PS, potentially increasing the utilization of grafts with infection risk. These findings require further validation in clinical trials. This study has several limitations. Firstly, although using clinically isolated strains (rather than commercial strains) improves clinical relevance, these isolates may not represent the full spectrum of antimicrobial resistance patterns. Secondly, the evaluation was limited to HC-A II PS (containing adenine phosphate, potassium citrate, sodium citrate, magnesium sulfate, and mannitol; pH 7.5 ± 0.1), leaving the efficacy in other preservation solutions (such as HTK and UW) unverified. Thirdly, despite standardization efforts, the requirement for fresh bacterial suspensions in each experiment lead to inherent variability in inoculum concentrations. Finally, viral and fungal graft contamination also could lead to severe donation related infections[ 21 – 23 ], highlighting the need for future research to develop comprehensive decontamination protocols that incorporate antiviral and antifungal agents to improve graft preservation. Conclusions Our study demonstrates that the combination of CAZ-AVI and ATM effectively inhibits MDR GNB in hypothermic PS within 3-hour, but the activity against Gram-positive bacteria remains limited. Notably, increasing the CAZ-AVI proportion could enhance efficacy, whereas higher ATM proportions provide little benefit. Further research is needed to evaluate this regimen’s efficacy in allografts and to explore its integration with machine perfusion systems for potent graft decontamination. Abbreviations ATM Aztreonam CAZ-AVI Ceftazidime-Avibactam CFU Colony-Forming Units CRAB Carbapenem-resistant Acinetobacter baumannii CREC Carbapenem-resistant Escherichia coli CRKP Carbapenem-resistant Klebsiella pneumoniae CRPA Carbapenem-resistant Pseudomonas aeruginosa MDRB Multidrug-resistant Bacteria MDR-GNB Multidrug-resistant Gram-negative Bacteria MRSA Methicillin-resistant Staphylococcus aureus PS Preservation Solution Declarations Ethics approval and consent to participate This study was conducted in accordance with the ethical principles of the Declaration of Helsinki. The Ethics Committee of Shanghai Changhai Hospital reviewed and approved the study protocol (Approval No. CHEC2021-024). Written informed consent was obtained from all participants prior to sample collection. Clinical trail number Not applicable. Consent for publication Not applicable. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare no competing interests. Funding This work was supported by the Project National Natural Science Foundation China (No.81700548), Sanhang Medical Talents Program (QH2022), Project for basic medical research of Shanghai Changhai hospital (2023PY08), the Medical Science and Technology Project (No.21QNY041), the ‘Deep Blue 123’ Medical Research Project (No.2020SLZ007) and the Changfeng Medical Talents Program (CY2023). Authors' contributions YZD and DQT contributed to the conceptualization, study design, and experimental execution. YHL and PZ assisted in data collection and analysis. YXW and KW provided critical bacterial strains and technical support. YFL , JHY , WYZ , and YHL participated in data validation and interpretation. MXS and LZ supervised the project, revised the manuscript for intellectual content, and approved the final version. All authors reviewed and agreed to the submitted manuscript. Acknowledgements We are deeply indebted to our Professor Youhua Zhu for his invaluable guidance, expert advice, and continuous encouragement throughout the project. His insightful feedback significantly improved the quality of our work. Declaration of competing interest None declared. References Yu X, Wang R, Peng W, Huang H, Liu G, Yang Q, et al. Incidence, distribution and clinical relevance of microbial contamination of preservation solution in deceased kidney transplant recipients: a retrospective cohort study from China. Clin Microbiol infection: official publication Eur Soc Clin Microbiol Infect Dis. 2019;25(5):595–600. 10.1016/j.cmi.2018.12.040 . Phillips BL, Chandak P, Uwechue R, van Nispen Tot Pannerden C, Hemsley C, Callaghan CJ. Microbial Contamination During Kidney Ex Vivo Normothermic Perfusion. Transplantation. 2018;102(4):e186–8. 10.1097/tp.0000000000002076 . Lau NS, Ly M, Dennis C, Toomath S, Huang JL, Huang J, et al. Microbial Contamination During Long-term Ex Vivo Normothermic Machine Perfusion of Human Livers. Transplantation. 2024;108(1):198–203. 10.1097/tp.0000000000004653 . Oriol I, Sabe N, Càmara J, Berbel D, Ballesteros MA, Escudero R, et al. The Impact of Culturing the Organ Preservation Fluid on Solid Organ Transplantation: A Prospective Multicenter Cohort Study. Open forum Infect Dis. 2019;6(6):ofz180. 10.1093/ofid/ofz180 . Sui M, Zheng N, Xu D, Li Y, Li Y, Pu S, et al. Colistin sulfate for decontamination of preservation fluid in kidney transplantation to decrease the incidence of donor-derived infections caused by multidrug-resistant Gram-negative bacteria. Transpl Infect disease: official J Transplantation Soc. 2022;24(3):e13820. 10.1111/tid.13820 . Patel G, Rana MM, Huprikar S. Multidrug-resistant bacteria in organ transplantation: an emerging threat with limited therapeutic options. Curr Infect Dis Rep. 2013;15(6):504–13. 10.1007/s11908-013-0371-z . Abdulqawi R, Saleh RA, Alameer RM, Aldakhil H, AlKattan KM, Almaghrabi RS, et al. Donor respiratory multidrug-resistant bacteria and lung transplantation outcomes. J Infect. 2024;88(2):139–48. 10.1016/j.jinf.2023.12.013 . Almohaya A, Fersovich J, Weyant RB, Fernández García OA, Campbell SM, Doucette K, et al. The impact of colonization by multidrug resistant bacteria on graft survival, risk of infection, and mortality in recipients of solid organ transplant: systematic review and meta-analysis. Clin Microbiol Infect. 2024;30(10):1228–43. 10.1016/j.cmi.2024.03.036 . Bush K, Bradford PA. Epidemiology of β-Lactamase-Producing Pathogens. Clin Microbiol Rev. 2020;33(2):37. 10.1128/cmr.00047-19 . Johnson DH, Cunha BA, Aztreonam. Med Clin N Am. 1995;79(4):733–43. 10.1016/s0025-7125(16)30036-0 . Paterson DL, Bonomo RA. Extended-spectrum beta-lactamases: a clinical update. Clin Microbiol Rev. 2005;18(4):657–86. 10.1128/cmr.18.4.657-686.2005 . Mauri C, Maraolo AE, Di Bella S, Luzzaro F, Principe L. The Revival of Aztreonam in Combination with Avibactam against Metallo-β-Lactamase-Producing Gram-Negatives: A Systematic Review of In Vitro Studies and Clinical Cases. Antibiot (Basel Switzerland). 2021;10(8). 10.3390/antibiotics10081012 . Wu Y, Chen J, Zhang G, Li J, Wang T, Kang W, et al. In-vitro activities of essential antimicrobial agents including aztreonam/avibactam, eravacycline, colistin and other comparators against carbapenem-resistant bacteria with different carbapenemase genes: A multi-centre study in China, 2021. Int J Antimicrob Agents. 2024;64(5):107341. 10.1016/j.ijantimicag.2024.107341 . Falcone M, Daikos GL, Tiseo G, Bassoulis D, Giordano C, Galfo V, et al. Efficacy of Ceftazidime-avibactam Plus Aztreonam in Patients With Bloodstream Infections Caused by Metallo-β-lactamase-Producing Enterobacterales. Clin Infect diseases: official publication Infect Dis Soc Am. 2021;72(11):1871–8. 10.1093/cid/ciaa586 . Yu W, Chen Y, Shen P, Ji J, Ying C, Liu Z, et al. Antibacterial Activity and Optimal Treatment of Ceftazidime-Avibactam and Aztreonam-Avibactam Against Bloodstream Infections Caused by Carbapenem-Resistant Klebsiella pneumoniae. Front Pharmacol. 2021;12:771910. 10.3389/fphar.2021.771910 . Wilhelm CM, Inamine E, Martins AF, Barth AL. Evaluation of Aztreonam and Ceftazidime/Avibactam Synergism against Klebsiella pneumoniae by MALDI-TOF MS. Antibiot (Basel Switzerland). 2023;12(6). 10.3390/antibiotics12061063 . Nakaminami H, Tajima M, Koishikawa K, Shiratori Y, Shioura M, Sasatsu M, et al. Development of effective antimicrobial cocktails to prevent bacterial contamination of allograft tissues under low temperature conditions. Interact Cardiovasc Thorac Surg. 2019;28(1):128–36. 10.1093/icvts/ivy209 . Liang H, Zhang P, Yu B, Liu Z, Pan L, He X, et al. Machine perfusion combined with antibiotics prevents donor-derived infections caused by multidrug-resistant bacteria. Am J Transplant. 2022;22(7):1791–803. 10.1111/ajt.17032 . Eshmuminov D, Mueller M, Brugger SD, Bautista Borrego L, Becker D, Hefti M, et al. Sources and prevention of graft infection during long-term ex situ liver perfusion. Transpl Infect disease: official J Transplantation Soc. 2021;23(4):e13623. 10.1111/tid.13623 . Slagter JS, Schurink CAM, Chong GM, Reinders MEJ, Porte RJ, Minnee RC. Limited Risk of Microbial Contamination During Hypothermic and Normothermic Machine Perfusion of Donor Kidneys. Transpl Infect disease: official J Transplantation Soc. 2025;e70019. 10.1111/tid.70019 . Hirsch HH, Randhawa PS. BK polyomavirus in solid organ transplantation-Guidelines from the American Society of Transplantation Infectious Diseases Community of Practice. Clin Transplant. 2019;33(9):e13528. 10.1111/ctr.13528 . Lu XX, Zhu WY, Wu GZ. Rabies virus transmission via solid organs or tissue allotransplantation. Infect Dis poverty. 2018;7(1):82. 10.1186/s40249-018-0467-7 . Stern S, Bezinover D, Rath PM, Paul A, Saner FH. Candida Contamination in Kidney and Liver Organ Preservation Solution: Does It Matter? J Clin Med. 2021;10(9). 10.3390/jcm10092022 . Additional Declarations No competing interests reported. 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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-7197658","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":503868769,"identity":"04703694-17e9-4e57-b4fa-49d3288cfa98","order_by":0,"name":"Yazhe Duan","email":"","orcid":"","institution":"Changhai Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yazhe","middleName":"","lastName":"Duan","suffix":""},{"id":503868770,"identity":"1e68f914-8ad0-41ba-9fab-cae50ae37617","order_by":1,"name":"Daqian Tang","email":"","orcid":"","institution":"Changhai Hospital","correspondingAuthor":false,"prefix":"","firstName":"Daqian","middleName":"","lastName":"Tang","suffix":""},{"id":503868772,"identity":"11c07e01-887c-468d-b46a-296979a6854c","order_by":2,"name":"Yuhong Li","email":"","orcid":"","institution":"Changhai Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yuhong","middleName":"","lastName":"Li","suffix":""},{"id":503868773,"identity":"96c210a8-3847-4710-8a97-74cd6867cdc3","order_by":3,"name":"Pei Zhang","email":"","orcid":"","institution":"Changhai Hospital","correspondingAuthor":false,"prefix":"","firstName":"Pei","middleName":"","lastName":"Zhang","suffix":""},{"id":503868774,"identity":"417e838e-129e-40fb-9eec-4abf7668f78d","order_by":4,"name":"Yuxiang Wan","email":"","orcid":"","institution":"Changhai Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yuxiang","middleName":"","lastName":"Wan","suffix":""},{"id":503868775,"identity":"feec6c68-8386-4c8c-a2aa-00827558f63a","order_by":5,"name":"Kang Wu","email":"","orcid":"","institution":"Changhai Hospital","correspondingAuthor":false,"prefix":"","firstName":"Kang","middleName":"","lastName":"Wu","suffix":""},{"id":503868776,"identity":"ad119db0-1bdc-4437-8b77-d930e7d0c1c3","order_by":6,"name":"Yanfeng Li","email":"","orcid":"","institution":"Changhai Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yanfeng","middleName":"","lastName":"Li","suffix":""},{"id":503868777,"identity":"17d6ac60-b5fd-4617-9e06-0b0729b2fe1c","order_by":7,"name":"Junhao Yu","email":"","orcid":"","institution":"Changhai Hospital","correspondingAuthor":false,"prefix":"","firstName":"Junhao","middleName":"","lastName":"Yu","suffix":""},{"id":503868778,"identity":"26068eee-6c3d-49ca-876b-743c4ed43330","order_by":8,"name":"Wenyu Zhao","email":"","orcid":"","institution":"Changhai Hospital","correspondingAuthor":false,"prefix":"","firstName":"Wenyu","middleName":"","lastName":"Zhao","suffix":""},{"id":503868779,"identity":"92893c2b-377b-4440-a108-342dc5b033f5","order_by":9,"name":"Yanhua Li","email":"","orcid":"","institution":"Changhai Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yanhua","middleName":"","lastName":"Li","suffix":""},{"id":503868780,"identity":"9fce4a7e-c4a2-426c-9e52-a0727f8e409a","order_by":10,"name":"Mingxing Sui","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYDACZhBhACIYGx9AxQyI1tIMU0pACxJgkyBKi2477+HXPAV37ObPSG6r/JmzLbGBvXmbBEPNHZxazA7zpVnOMHiWvOFGYttt3m23Ext4jpVJMBx7hkcLj5nBB4PDyQYSQC2MIC0SOWYSjA2H8WtJAGqRn5HYVvgTpEX+DUEtxg+AttgxAB3GAHaYBA9hWxhnGBxOMDjzsFkaqMW4jSet2CLhGB4t588Yf+b5c9hevj394Uegw2T72Q9vvPGhBrcWBmh0JDbAuSAiAZ8GYPx/ABL2+NWMglEwCkbBiAYAhcZY2GiHdGcAAAAASUVORK5CYII=","orcid":"","institution":"Changhai Hospital","correspondingAuthor":true,"prefix":"","firstName":"Mingxing","middleName":"","lastName":"Sui","suffix":""},{"id":503868781,"identity":"ff29fa91-1f7e-464b-9f93-1ebdae723cbd","order_by":11,"name":"Li Zeng","email":"","orcid":"","institution":"Changhai Hospital","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Zeng","suffix":""}],"badges":[],"createdAt":"2025-07-23 14:53:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7197658/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7197658/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89692272,"identity":"f72a816d-49b9-4b50-8e83-53a6a54d0003","added_by":"auto","created_at":"2025-08-22 17:07:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":208708,"visible":true,"origin":"","legend":"\u003cp\u003eAntibacterial efficacy of CAZ-AVI/ATM against CRKP after 3-hour decontamination at 0–4°C. Results are presented as differences in CFU between treatment groups and the blank control across four different ratios and concentrations. \u003cstrong\u003eCRKP\u003c/strong\u003e, carbapenem-resistant \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e; \u003cstrong\u003eCFU\u003c/strong\u003e, colony-forming units.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7197658/v1/95b50a05a0c9b805c472d608.png"},{"id":89691901,"identity":"6b3550fe-f0f2-4318-b371-dff16d148d1b","added_by":"auto","created_at":"2025-08-22 16:59:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":440516,"visible":true,"origin":"","legend":"\u003cp\u003eAntibacterial efficacy of CAZ-AVI Combined with ATM against \u003cstrong\u003eCRPA (A), CREC (B), and CRAB (C) \u003c/strong\u003eafter 3-hour decontamination at 0–4°C. Results are presented as the differences in CFU between treatment groups and the blank control, evaluated across three ratios and different concentrations. \u003cstrong\u003eCRPA\u003c/strong\u003e, carbapenem-resistant \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e; \u003cstrong\u003eCREC\u003c/strong\u003e, carbapenem-resistant \u003cem\u003eEscherichia coli\u003c/em\u003e; \u003cstrong\u003eCRAB\u003c/strong\u003e, carbapenem-resistant \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e; \u003cstrong\u003eCFU\u003c/strong\u003e, colony-forming units.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7197658/v1/c1b556a48585953cba386ed6.png"},{"id":89693174,"identity":"0977802a-2e37-4398-91a2-46dc0148926a","added_by":"auto","created_at":"2025-08-22 17:23:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":183757,"visible":true,"origin":"","legend":"\u003cp\u003eAntibacterial efficacy of CAZ-AVI/ATM against MRSA after 3-hour decontamination at 0-4°C. Results are presented as the differences in CFU between treatment groups and the blank control across 4 different ratios and concentrations. MRSA, methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e; CFU, colony-forming units.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7197658/v1/3ec68e06bfa40887f12c2abf.png"},{"id":93074838,"identity":"93598087-f283-41aa-86c4-78dd4c348268","added_by":"auto","created_at":"2025-10-08 18:41:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1554426,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7197658/v1/db7b7f9e-2954-4f32-813a-9466c3893ce7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Study on the Antibacterial Efficacy of Ceftazidime-Avibactam Combined with Aztreonam against Multidrug-Resistant Bacteria in Hypothermic Organ Preservation Solution","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOrgan transplantation is a critical treatment for end-stage organ failure, with numerous procedures performed annually to save patients\u0026rsquo; lives. However, allografts may be contaminated by bacteria originating from donors or during the acquisition, preservation or transportation processes[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. To prevent bacterial transmission to recipients, the addition of antimicrobial agents to preservation solution (PS) for decontamination has been adopted by transplant centers worldwide[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Nevertheless, multidrug-resistant bacteria (MDRB) pose significant challenges and the MDRB related graft contamination may severely compromises transplant outcomes[\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Therefore, developing novel antimicrobial strategies to enhance decontamination efficiency against MDRB is imperative.\u003c/p\u003e\u003cp\u003eAvibactam (AVI), a novel β-lactamase inhibitor, is effective against class A, C, and D β-lactamases (e.g., KPC, AmpC, and OXA-48) but exhibits no activity against class B metallo-β-lactamases (such as NDM and VIM)\u003csup\u003e[9]\u003c/sup\u003e. Aztreonam (ATM), a monobactam antibiotic, demonstrates intrinsic stability against NDM\u003csup\u003e[10]\u003c/sup\u003e and is easily hydrolyzed by extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases\u003csup\u003e[11, 12]\u003c/sup\u003e. The combination of these agents (CAZ-AVI/ATM) could enhance the antimicrobial effect against MDRB due to their complementary mechanisms[\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe primary objective of this study is to investigate the bactericidal efficacy of this combination against MDRB in low-temperature PS, which remains undocumented. We will investigate whether the synergistic application of drugs at varying ratios and concentrations significantly reduces bacterial contamination in PS and determine the optimal drug combination for effective decontamination.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMaterials\u003c/h2\u003e\u003cp\u003eCAZ-AVI for injection (250 mg; CAZ 200 mg\u0026thinsp;+\u0026thinsp;AVI 50 mg) was obtained from Qilu Pharmaceutical Co., Ltd. (Jinan, China). ATM for injection (1 g) was sourced from Luoxin Pharmaceutical Co., Ltd. (Linyi, China). The organ preservation solution, hypertonic citrate adenine solution II (HC-A II), was acquired from Shanghai Haini Pharmaceutical Co., Ltd. (Shanghai, China). Additional materials included: a DensiCHEK-plus densitometer (BioM\u0026eacute;rieux, France), Columbia blood agar plates (Φ90 mm; Kemajia Shanghai Microbe Technology Co., Ltd., Shanghai, China), disposable plastic spreaders (BS-PS-A; Labgic Technology Co., Ltd., Beijing, China), and sterile 96-well cell plates (Corning Life Sciences Co., Ltd., Suzhou, China).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eBacterial Strains and Culture Conditions\u003c/h3\u003e\n\u003cp\u003eFive clinically representative MDRB were selected for this study: carbapenem-resistant \u003cem\u003eKlebsiella\u003c/em\u003e pneumoniae (CRKP), carbapenem-resistant \u003cem\u003ePseudomonas\u003c/em\u003e aeruginosa (CRPA), carbapenem-resistant \u003cem\u003eEscherichia\u003c/em\u003e coli (CREC), carbapenem-resistant \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii (CRAB), and methicillin-resistant \u003cem\u003eStaphylococcus\u003c/em\u003e aureus (MRSA). Clinical isolates were obtained from inpatients at Shanghai Changhai Hospital. All bacterial strains were isolated and cultured in the microbiology laboratory following the National Clinical Laboratory Practice Guidelines (4th edition). Species identification was performed using the MALDI Biotyper system (Bruker Daltonik, Germany). Antibiotic susceptibility testing was conducted via the disk diffusion method. After the bacteria were isolated and cultured from specimens, individual bacterial colonies were inoculated into the designated broth media, all samples underwent genetic testing by fluorescent PCR to determine bacterial genotypes/phenotypes and detect antibiotic resistance genes (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Prior to experiments, bacterial stocks were preserved at \u0026minus;\u0026thinsp;80\u0026deg;C. For reactivation, one frozen strain was subcultured twice on Columbia blood agar plates. A single colony was then aseptically suspended in PS, and the turbidity was adjusted to 0.5 McFarland standard using a densitometer.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eResults of antibiotic resistance genes detection for various strains\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStrains\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eResistance Gene 1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eResistance Gene 2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eResistance Gene 3\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCRKP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCTX-M\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eKPC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNDM\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCRPA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCREC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCTX-M\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNDM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCRAB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOXA-23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMRSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eCRKP\u003c/em\u003e carbapenem-resistant \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, \u003cem\u003eCRPA\u003c/em\u003e carbapenem-resistant \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, \u003cem\u003eCREC\u003c/em\u003e carbapenem-resistant \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eCRAB\u003c/em\u003e carbapenem-resistant \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e, \u003cem\u003eMRSA\u003c/em\u003e methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eCTX-M\u003c/em\u003e Cefotaxime-hydrolyzing β-lactamase-Munich, \u003cem\u003eKPC\u003c/em\u003e Klebsiella pneumoniae carbapenemase, \u003cem\u003eNDM\u003c/em\u003e New Delhi metallo-β-lactamase, \u003cem\u003eOXA-23\u003c/em\u003e Oxacillin-hydrolyzing β-lactamase-23\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.3. Definition of CAZ-AVI/ATM Ratios and Concentrations\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe baseline concentration was defined as 250 mg/L CAZ-AVI\u0026thinsp;+\u0026thinsp;1 g/L ATM, and this ratio was considered as 1:1. Four drug ratios were established: 1:1, 1:2, 1:4, and 2:1. For each ratio, four concentration gradients were tested: 0.5\u0026times;, 1\u0026times;, 2\u0026times;, and 4\u0026times; the baseline concentration. Aliquots (50 \u0026micro;l) of each antibiotic solution were dispensed into 96-well plates and pre-chilled at 0\u0026ndash;4\u0026deg;C for 24 hours prior to experimentation. The complete matrix of CAZ-AVI/ATM ratios and concentrations is detailed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe ratios and concentrations of CAZ-AVI/ATM\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCAZ-AVI : ATM\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.5\u0026times;\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u0026times;\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2\u0026times;\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4\u0026times;\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1 : 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e125mg/L\u0026thinsp;+\u0026thinsp;0.5g/L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e250mg/L\u0026thinsp;+\u0026thinsp;1.0g/L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e500mg/L\u0026thinsp;+\u0026thinsp;2.0g/L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1000mg/L\u0026thinsp;+\u0026thinsp;4.0g/L\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1 : 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e125mg/L\u0026thinsp;+\u0026thinsp;1.0g/L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e250mg/L\u0026thinsp;+\u0026thinsp;2.0g/L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e500mg/L\u0026thinsp;+\u0026thinsp;4.0g/L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1000mg/L\u0026thinsp;+\u0026thinsp;8.0g/L\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1 : 4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e125mg/L\u0026thinsp;+\u0026thinsp;2.0g/L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e250mg/L\u0026thinsp;+\u0026thinsp;4.0g/L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e500mg/L\u0026thinsp;+\u0026thinsp;8.0g/L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1000mg/L\u0026thinsp;+\u0026thinsp;16.0g/L\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2 : 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e250mg/L\u0026thinsp;+\u0026thinsp;0.5g/L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e500mg/L\u0026thinsp;+\u0026thinsp;1.0g/L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1000mg/L\u0026thinsp;+\u0026thinsp;2.0g/L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2000mg/L\u0026thinsp;+\u0026thinsp;4.0g/L\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eEvaluation of MDRB Decontamination by CAZ-AVI/ATM Under 3-Hour Cold Storage Conditions\u003c/h3\u003e\n\u003cp\u003eBlank controls (antibiotic-free) and treatment groups were established with 6\u0026ndash;9 replicates per condition. Bacterial suspensions were serially diluted with PS, and 50 \u0026micro;l aliquots were inoculated into pre-chilled 96-well plates. After thorough mixing, plates were maintained at 0\u0026ndash;4\u0026deg;C for 3 hours to simulate clinical graft preservation conditions, yielding a final bacterial concentration of 1.5 \u0026times; 10⁴ CFU/ml. After decontamination,10 \u0026micro;l aliquots from each well were plated onto Columbia blood agar plates, uniformly spread using sterile plastic spreaders (BS-PS-A), and incubated at 37\u0026deg;C for 20\u0026ndash;24 hours. Colony-forming units (CFU) were quantified, and bacterial identity was confirmed by MALDI-TOF mass spectrometry. The optimal antibiotic ratio and minimum effective concentration were determined through statistical comparison of CFU reduction between treatment groups and blank controls.\u003c/p\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using IBM SPSS Statistics 24 and GraphPad Prism 9.5. Continuous data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Based on normality (Shapiro-Wilk test) and homogeneity of variance (Levene's test), we applied: One-way ANOVA with LSD test (normal distribution, equal variance), Welch ANOVA with Games-Howell test (normal distribution, unequal variance), Kruskal-Wallis test with Dunn's correction (non-normal distribution). A two-tailed \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eRatio Selection of CAZ-AVI/ATM\u003c/h2\u003e\n \u003cp\u003eThe antimicrobial efficacy of CAZ-AVI/ATM against CRKP was illustrated in Fig.\u0026nbsp;1. All tested ratios exhibited concentration-dependent bactericidal activity, with the 1:1 ratio demonstrating statistically significant reductions in colony counts compared to blank controls at concentrations\u0026thinsp;\u0026ge;\u0026thinsp;1\u0026times; (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.016 for 1\u0026times;, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.009 for 2\u0026times;, and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002 for 4\u0026times;). The 1:2 and 1:4 ratios required higher concentrations (2\u0026times; and 1\u0026times;, respectively) to achieve statistical significance (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.022 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.039, respectively). Notably, the 2:1 ratio showed a better synergistic effect, exhibiting significant bacterial reduction even at the 0.5\u0026times; concentration (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002).\u003c/p\u003e\n \u003cp\u003eThese findings suggest that increasing the proportion of CAZ-AVI enhances synergistic antimicrobial activity more effectively than increasing ATM. Based on these results, the subsequent experiments focused on the 2:1, 1:1 and 1:4 groups to further investigate this synergistic pattern.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eAntibacterial Efficacy of CAZ-AVI/ ATM Against MDR-GNB Under 3-Hour Hypothermic Conditions\u003c/h3\u003e\n\u003cp\u003eFor CRPA (Fig.\u0026nbsp;2A), both the 1:1 and 1:4 groups required\u0026thinsp;\u0026ge;\u0026thinsp;2\u0026times; concentrations to demonstrate statistically significant differences compared to the blank control (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.009 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001, respectively), while the 2:1 group showed significant differences at the 1\u0026times; concentration (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.022). For CREC (Fig.\u0026nbsp;2B), the 1:4 and 2:1 groups exhibited significant bactericidal activity at the 0.5\u0026times; concentration (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.017 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively), the 1:1 group required\u0026thinsp;\u0026ge;\u0026thinsp;1\u0026times; concentrations to achieve comparable effects (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). For CRAB (Fig. 2C), both the 1:1 and 2:1 groups showed significant CFU reduction versus blank controls at the lowest concentration (0.5\u0026times;; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.034, respectively), whereas the 1:4 group failed to demonstrate statistical significance at either 0.5\u0026times; or 1\u0026times; (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.356 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.117, respectively).\u003c/p\u003e\n\u003ch3\u003eAntibacterial Efficacy of CAZ-AVI/ATM Against MRSA Under 3-Hour Hypothermic Conditions\u003c/h3\u003e\n\u003cp\u003eAs shown in Fig.\u0026nbsp;3, no statistically significant differences were observed among groups when using 1:1, 1:2, or 1:4 ratios (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.181, 0.697, and 0.112, respectively). However, when the ratio was adjusted to 2:1 and the concentration increased to \u0026ge;\u0026thinsp;2\u0026times;, statistically significant differences were achieved compared to the blank controls (both \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe antimicrobial efficacy evaluation under hypothermic conditions presents unique pharmacological challenges. Existing evidence demonstrates that minimal bactericidal concentration (MBC) at 4\u0026deg;C may be hundreds to thousands of times higher than standard MBC values[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], underscoring the critical need for condition-specific antimicrobial assessments. This study evaluated the CAZ-AVI/ATM efficacy against five clinically significant MDRB strains during a 3-hour low-temperature (0\u0026ndash;4\u0026deg;C) decontamination protocol using HC-A II PS. The findings established both the optimal drug ratio and the minimal effective concentration required for reducing bacterial contamination under clinically relevant preservation conditions.\u003c/p\u003e\u003cp\u003eThe preliminary investigation of CAZ-AVI/ATM against CRKP (Fig.\u0026nbsp;1) revealed that 1:1 or ATM-dominant ratios (1:2, and 1:4) achieved significant bacterial growth inhibition at 1\u0026times; or 2\u0026times; concentrations, whereas the CAZ-AVI-dominant ratio (2:1) demonstrated satisfactory bactericidal effects at 0.5\u0026times; concentration, suggesting enhanced synergy with higher CAZ-AVI proportions.\u003c/p\u003e\u003cp\u003eIn subsequent experiments comparing the bactericidal efficacy of three ratios (1:1, 1:4, and 2:1) against CRPA, CREC, and CRAB, the results demonstrated that the CAZ-AVI-dominant ratio achieved effective inhibition at only 0.5\u0026times; or 1\u0026times; concentration while the 1:1 and ATM-dominant ratio required\u0026thinsp;\u0026ge;\u0026thinsp;2\u0026times; concentration to inhibit bacterial growth. Specially, the ATM-dominant ratio exhibited inferior bactericidal efficacy against CRAB compared to the 1:1 at the same concentration. We further investigated the bactericidal efficacy against MRSA. The results were not satisfactory and only the CAZ-AVI-dominant ratio (2:1) exhibited bactericidal effects at higher concentrations (2\u0026times; and 4\u0026times;) (Fig.\u0026nbsp;3). These comprehensive results demonstrate that CAZ-AVI/ATM effectively inhibit these MDR GNB in PS under the condition of clinic decontamination procedure, with synergy enhanced by increasing CAZ-AVI ratio but limited enhancement by higher ATM ratio. Furthermore, CAZ-AVI has limited activity against Gram-positive organisms; however, sufficiently high concentration may achieve antibacterial effects. Considering the cost-effectiveness and antimicrobial efficacy, our findings identify the 2:1 ratio as optimal (compared with 1:1, 1:2, and 1:4), though minimum effective concentrations vary slightly among pathogens as detailed in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cimg 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\" width=\"584\" height=\"497\"\u003e\u003c/p\u003e\u003cp\u003eThe PS decontamination regimen does not rely only on antibiotics, and soaking and perfusion can also help remove pathogens. Recent evidence suggests that machine perfusion could enhance bacterial clearance through its closed-loop system, which maintains higher antibiotic concentrations within the organ while providing mechanical flushing[\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Based on this study, we recommend adding 500mg CAZ-AVI and 1g ATM per liter of PS, with supplemental MRSA-targeted antibiotics if the donor was at high risk. Combining this protocol with machine perfusion may further improve bacterial clearance in contaminated PS, potentially increasing the utilization of grafts with infection risk. These findings require further validation in clinical trials.\u003c/p\u003e\u003cp\u003eThis study has several limitations. Firstly, although using clinically isolated strains (rather than commercial strains) improves clinical relevance, these isolates may not represent the full spectrum of antimicrobial resistance patterns. Secondly, the evaluation was limited to HC-A II PS (containing adenine phosphate, potassium citrate, sodium citrate, magnesium sulfate, and mannitol; pH 7.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1), leaving the efficacy in other preservation solutions (such as HTK and UW) unverified. Thirdly, despite standardization efforts, the requirement for fresh bacterial suspensions in each experiment lead to inherent variability in inoculum concentrations. Finally, viral and fungal graft contamination also could lead to severe donation related infections[\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], highlighting the need for future research to develop comprehensive decontamination protocols that incorporate antiviral and antifungal agents to improve graft preservation.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur study demonstrates that the combination of CAZ-AVI and ATM effectively inhibits MDR GNB in hypothermic PS within 3-hour, but the activity against Gram-positive bacteria remains limited. Notably, increasing the CAZ-AVI proportion could enhance efficacy, whereas higher ATM proportions provide little benefit. Further research is needed to evaluate this regimen\u0026rsquo;s efficacy in allografts and to explore its integration with machine perfusion systems for potent graft decontamination.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eATM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAztreonam\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCAZ-AVI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCeftazidime-Avibactam\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCFU\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eColony-Forming Units\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCRAB\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCarbapenem-resistant\u0026nbsp;Acinetobacter baumannii\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCREC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCarbapenem-resistant\u0026nbsp;Escherichia coli\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCRKP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCarbapenem-resistant\u0026nbsp;Klebsiella pneumoniae\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCRPA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCarbapenem-resistant\u0026nbsp;Pseudomonas aeruginosa\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMDRB\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMultidrug-resistant Bacteria\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMDR-GNB\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMultidrug-resistant Gram-negative Bacteria\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMRSA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMethicillin-resistant\u0026nbsp;Staphylococcus aureus\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePreservation Solution\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003eThis study was conducted in accordance with the ethical principles of the Declaration of Helsinki. The Ethics Committee of Shanghai Changhai Hospital reviewed and approved the study protocol (Approval No. CHEC2021-024). Written informed consent was obtained from all participants prior to sample collection.\u003c/p\u003e\n\u003ch2\u003eClinical trail number\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the Project National Natural Science Foundation China (No.81700548), Sanhang Medical Talents Program (QH2022), Project for basic medical research of Shanghai Changhai hospital (2023PY08), the Medical Science and Technology Project (No.21QNY041), the \u0026lsquo;Deep Blue 123\u0026rsquo; Medical Research Project (No.2020SLZ007) and the Changfeng Medical Talents Program (CY2023).\u003c/p\u003e\n\u003ch2\u003eAuthors\u0026apos; contributions\u003c/h2\u003e\n\u003cp\u003e\u003cstrong\u003eYZD\u003c/strong\u003e and \u003cstrong\u003eDQT\u003c/strong\u003e contributed to the conceptualization, study design, and experimental execution. \u003cstrong\u003eYHL\u003c/strong\u003e and \u003cstrong\u003ePZ\u003c/strong\u003e assisted in data collection and analysis. \u003cstrong\u003eYXW\u003c/strong\u003e and \u003cstrong\u003eKW\u003c/strong\u003e provided critical bacterial strains and technical support. \u003cstrong\u003eYFL\u003c/strong\u003e, \u003cstrong\u003eJHY\u003c/strong\u003e, \u003cstrong\u003eWYZ\u003c/strong\u003e, and \u003cstrong\u003eYHL\u003c/strong\u003e participated in data validation and interpretation. \u003cstrong\u003eMXS\u0026nbsp;\u003c/strong\u003eand \u003cstrong\u003eLZ\u003c/strong\u003e supervised the project, revised the manuscript for intellectual content, and approved the final version. All authors reviewed and agreed to the submitted manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eWe are deeply indebted to our Professor Youhua Zhu for his invaluable guidance, expert advice, and continuous encouragement throughout the project. His insightful feedback significantly improved the quality of our work.\u003c/p\u003e\n\u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e\n\u003cp\u003eNone declared.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYu X, Wang R, Peng W, Huang H, Liu G, Yang Q, et al. Incidence, distribution and clinical relevance of microbial contamination of preservation solution in deceased kidney transplant recipients: a retrospective cohort study from China. Clin Microbiol infection: official publication Eur Soc Clin Microbiol Infect Dis. 2019;25(5):595\u0026ndash;600. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cmi.2018.12.040\u003c/span\u003e\u003cspan address=\"10.1016/j.cmi.2018.12.040\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePhillips BL, Chandak P, Uwechue R, van Nispen Tot Pannerden C, Hemsley C, Callaghan CJ. Microbial Contamination During Kidney Ex Vivo Normothermic Perfusion. Transplantation. 2018;102(4):e186\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1097/tp.0000000000002076\u003c/span\u003e\u003cspan address=\"10.1097/tp.0000000000002076\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLau NS, Ly M, Dennis C, Toomath S, Huang JL, Huang J, et al. Microbial Contamination During Long-term Ex Vivo Normothermic Machine Perfusion of Human Livers. Transplantation. 2024;108(1):198\u0026ndash;203. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1097/tp.0000000000004653\u003c/span\u003e\u003cspan address=\"10.1097/tp.0000000000004653\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOriol I, Sabe N, C\u0026agrave;mara J, Berbel D, Ballesteros MA, Escudero R, et al. The Impact of Culturing the Organ Preservation Fluid on Solid Organ Transplantation: A Prospective Multicenter Cohort Study. Open forum Infect Dis. 2019;6(6):ofz180. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/ofid/ofz180\u003c/span\u003e\u003cspan address=\"10.1093/ofid/ofz180\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSui M, Zheng N, Xu D, Li Y, Li Y, Pu S, et al. Colistin sulfate for decontamination of preservation fluid in kidney transplantation to decrease the incidence of donor-derived infections caused by multidrug-resistant Gram-negative bacteria. Transpl Infect disease: official J Transplantation Soc. 2022;24(3):e13820. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/tid.13820\u003c/span\u003e\u003cspan address=\"10.1111/tid.13820\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePatel G, Rana MM, Huprikar S. Multidrug-resistant bacteria in organ transplantation: an emerging threat with limited therapeutic options. Curr Infect Dis Rep. 2013;15(6):504\u0026ndash;13. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s11908-013-0371-z\u003c/span\u003e\u003cspan address=\"10.1007/s11908-013-0371-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAbdulqawi R, Saleh RA, Alameer RM, Aldakhil H, AlKattan KM, Almaghrabi RS, et al. Donor respiratory multidrug-resistant bacteria and lung transplantation outcomes. J Infect. 2024;88(2):139\u0026ndash;48. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jinf.2023.12.013\u003c/span\u003e\u003cspan address=\"10.1016/j.jinf.2023.12.013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlmohaya A, Fersovich J, Weyant RB, Fern\u0026aacute;ndez Garc\u0026iacute;a OA, Campbell SM, Doucette K, et al. The impact of colonization by multidrug resistant bacteria on graft survival, risk of infection, and mortality in recipients of solid organ transplant: systematic review and meta-analysis. Clin Microbiol Infect. 2024;30(10):1228\u0026ndash;43. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cmi.2024.03.036\u003c/span\u003e\u003cspan address=\"10.1016/j.cmi.2024.03.036\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBush K, Bradford PA. Epidemiology of β-Lactamase-Producing Pathogens. Clin Microbiol Rev. 2020;33(2):37. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/cmr.00047-19\u003c/span\u003e\u003cspan address=\"10.1128/cmr.00047-19\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJohnson DH, Cunha BA, Aztreonam. Med Clin N Am. 1995;79(4):733\u0026ndash;43. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/s0025-7125(16)30036-0\u003c/span\u003e\u003cspan address=\"10.1016/s0025-7125(16)30036-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePaterson DL, Bonomo RA. Extended-spectrum beta-lactamases: a clinical update. Clin Microbiol Rev. 2005;18(4):657\u0026ndash;86. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/cmr.18.4.657-686.2005\u003c/span\u003e\u003cspan address=\"10.1128/cmr.18.4.657-686.2005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMauri C, Maraolo AE, Di Bella S, Luzzaro F, Principe L. The Revival of Aztreonam in Combination with Avibactam against Metallo-β-Lactamase-Producing Gram-Negatives: A Systematic Review of In Vitro Studies and Clinical Cases. Antibiot (Basel Switzerland). 2021;10(8). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/antibiotics10081012\u003c/span\u003e\u003cspan address=\"10.3390/antibiotics10081012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWu Y, Chen J, Zhang G, Li J, Wang T, Kang W, et al. In-vitro activities of essential antimicrobial agents including aztreonam/avibactam, eravacycline, colistin and other comparators against carbapenem-resistant bacteria with different carbapenemase genes: A multi-centre study in China, 2021. Int J Antimicrob Agents. 2024;64(5):107341. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ijantimicag.2024.107341\u003c/span\u003e\u003cspan address=\"10.1016/j.ijantimicag.2024.107341\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFalcone M, Daikos GL, Tiseo G, Bassoulis D, Giordano C, Galfo V, et al. Efficacy of Ceftazidime-avibactam Plus Aztreonam in Patients With Bloodstream Infections Caused by Metallo-β-lactamase-Producing Enterobacterales. Clin Infect diseases: official publication Infect Dis Soc Am. 2021;72(11):1871\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/cid/ciaa586\u003c/span\u003e\u003cspan address=\"10.1093/cid/ciaa586\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYu W, Chen Y, Shen P, Ji J, Ying C, Liu Z, et al. Antibacterial Activity and Optimal Treatment of Ceftazidime-Avibactam and Aztreonam-Avibactam Against Bloodstream Infections Caused by Carbapenem-Resistant Klebsiella pneumoniae. Front Pharmacol. 2021;12:771910. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fphar.2021.771910\u003c/span\u003e\u003cspan address=\"10.3389/fphar.2021.771910\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWilhelm CM, Inamine E, Martins AF, Barth AL. Evaluation of Aztreonam and Ceftazidime/Avibactam Synergism against Klebsiella pneumoniae by MALDI-TOF MS. Antibiot (Basel Switzerland). 2023;12(6). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/antibiotics12061063\u003c/span\u003e\u003cspan address=\"10.3390/antibiotics12061063\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNakaminami H, Tajima M, Koishikawa K, Shiratori Y, Shioura M, Sasatsu M, et al. Development of effective antimicrobial cocktails to prevent bacterial contamination of allograft tissues under low temperature conditions. Interact Cardiovasc Thorac Surg. 2019;28(1):128\u0026ndash;36. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/icvts/ivy209\u003c/span\u003e\u003cspan address=\"10.1093/icvts/ivy209\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiang H, Zhang P, Yu B, Liu Z, Pan L, He X, et al. Machine perfusion combined with antibiotics prevents donor-derived infections caused by multidrug-resistant bacteria. Am J Transplant. 2022;22(7):1791\u0026ndash;803. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/ajt.17032\u003c/span\u003e\u003cspan address=\"10.1111/ajt.17032\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEshmuminov D, Mueller M, Brugger SD, Bautista Borrego L, Becker D, Hefti M, et al. Sources and prevention of graft infection during long-term ex situ liver perfusion. Transpl Infect disease: official J Transplantation Soc. 2021;23(4):e13623. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/tid.13623\u003c/span\u003e\u003cspan address=\"10.1111/tid.13623\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSlagter JS, Schurink CAM, Chong GM, Reinders MEJ, Porte RJ, Minnee RC. Limited Risk of Microbial Contamination During Hypothermic and Normothermic Machine Perfusion of Donor Kidneys. Transpl Infect disease: official J Transplantation Soc. 2025;e70019. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/tid.70019\u003c/span\u003e\u003cspan address=\"10.1111/tid.70019\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHirsch HH, Randhawa PS. BK polyomavirus in solid organ transplantation-Guidelines from the American Society of Transplantation Infectious Diseases Community of Practice. Clin Transplant. 2019;33(9):e13528. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/ctr.13528\u003c/span\u003e\u003cspan address=\"10.1111/ctr.13528\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLu XX, Zhu WY, Wu GZ. Rabies virus transmission via solid organs or tissue allotransplantation. Infect Dis poverty. 2018;7(1):82. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s40249-018-0467-7\u003c/span\u003e\u003cspan address=\"10.1186/s40249-018-0467-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStern S, Bezinover D, Rath PM, Paul A, Saner FH. Candida Contamination in Kidney and Liver Organ Preservation Solution: Does It Matter? J Clin Med. 2021;10(9). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/jcm10092022\u003c/span\u003e\u003cspan address=\"10.3390/jcm10092022\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Organ transplantation, Multidrug-resistant bacteria, Ceftazidime-avibactam, Aztreonam, Organ preservation solution","lastPublishedDoi":"10.21203/rs.3.rs-7197658/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7197658/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eThe use of antimicrobial agents in the preservation solution (PS) could prevent the bacterial transmission to recipients in organ transplantation. However, antibiotics may exhibit different decontamination efficacy in the hypothermic PS. This study aimed to evaluate the antibacterial activity of ceftazidime-avibactam (CAZ-AVI) combined with aztreonam (ATM) against multidrug-resistant bacteria (MDRB) at varying ratios and concentrations during 3-hour, 0\u0026ndash;4℃ PS decontamination.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eFor CRKP and CREC, the 2:1 ratio at 0.5\u0026times; concentration (250 mg/L CAZ-AVI\u0026thinsp;+\u0026thinsp;0.5 g/L ATM) showed significant antibacterial effects (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively). For CRPA, efficacy was observed at the 1\u0026times; concentration with a 2:1 ratio (500 mg/L CAZ-AVI\u0026thinsp;+\u0026thinsp;1.0 g/L ATM; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.022), while for CRAB, the 1:1 ratio at 0.5\u0026times; concentration (125 mg/L CAZ-AVI\u0026thinsp;+\u0026thinsp;0.5 g/L ATM; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was effective. The combination was only effective against MRSA at high concentrations (1000 mg/L CAZ-AVI\u0026thinsp;+\u0026thinsp;2.0 g/L ATM; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThe combination of CAZ-AVI and ATM effectively decontaminates MDR Gram-negative bacteria in PS. The 2:1 ratio at baseline concentration is recommended for clinical use, with potential escalation of CAZ-AVI concentration if needed.\u003c/p\u003e","manuscriptTitle":"Study on the Antibacterial Efficacy of Ceftazidime-Avibactam Combined with Aztreonam against Multidrug-Resistant Bacteria in Hypothermic Organ Preservation Solution","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-22 16:58:55","doi":"10.21203/rs.3.rs-7197658/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"50152e83-43c5-49f6-b1bd-5a740d625e63","owner":[],"postedDate":"August 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-08T18:40:49+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-22 16:58:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7197658","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7197658","identity":"rs-7197658","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}