Development and Clinical Application of a PCR-UV Assay for Detection of Carbapenem Resistant Acinetobacter baumannii in Bloodstream infections 

Development and Clinical Application of a PCR-UV Assay for Detection of Carbapenem Resistant Acinetobacter baumannii in Bloodstream infections | 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 Development and Clinical Application of a PCR-UV Assay for Detection of Carbapenem Resistant Acinetobacter baumannii in Bloodstream infections Lin Yu, Jingzhi Zhang, Ze Liu, Siyi Liu, Yandong Zhang, Ziman Wu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4723438/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Oct, 2025 Read the published version in Microbial Cell Factories → Version 1 posted 12 You are reading this latest preprint version Abstract Objective: Bloodstream infections caused by carbapenem-resistant Acinetobacter baumannii (CRAB) are a significant public health concern, with high morbidity and mortality rates. The detection and identification of CRAB is essential for early diagnosis and treatment. Hence, a rapid and economical CRAB-associated Bloodstream infections(BSIs) detection method is of urgent need. Methods: A tripling PCR-UV reaction system was designed for detecting the antibiotic resistance gene OXA23, OXA51 and AB-specific gene. The specificity of the primers , limit of detection (LOD), reproducibility, and accuracy of the assay were evaluated. The PCR products were analyzed using UV and ImageJ analysis directly, which provided a quickly interpretation of the results. Furthermore, the established assay was validated on clinical isolates and compared with blood culture and drug sensitivity tests. Results: The tripling PCR-UV method established in this study demonstrated strong primer specificity, distinguishing CRAB among 23 common clinical pathogens. The results of this PCR method have been validated by electrophoresis for good accuracy and reproducibility, with a Limit of Detection (LOD) of 3.0 x 10 -1 ng/uL. Meanwhile, the optimal annealing temperature for the triple method was optimized to 56.4 ℃. The result of PCR amplification could be judged by the result of the gray value of the tube to be tested / the gray value of the blank control of the same batch. The ratio>1.3 is CRAB, the ratio between 1.1-1.3 is carbapenem-sensitive Acinetobacter baumannii (CSAB), the ratio<1.1 is negative result. When applied to detect 30 patients with BSIs of AB, the results were consistent with clinical blood culture identification and drug sensitivity tests. Conclusion: The tripling PCR-UV assay developed in this study is a UV-visual, rapid, and cost-effective method for the detection of Acinetobacter baumannii (AB ) and identification of CRAB in bloodstream infections. The assay could be particularly useful in grassroots units where expensive molecular instruments are not readily available and could help in the diagnosis and treatment of CRAB infections in BSIs. carbapenem-resistant Acinetobacter baumannii(CRAB) carbapenem-resistance gene Bloodstream infections(BSIs) PCR-UV assay Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Acinetobacter baumannii (AB), a gram-positive bacterium, is a significant human pathogen that has the ability to cause a wide array of severe hospital and community-acquired infections, particularly BSIs [1] . BSIs is a major concern in hospitals worldwide, commonly known as nosocomial bloodstream infections (NBSI). AB is one of the most important opportunistic pathogens causing NBSI due to its natural and acquired resistance to antibiotics, particularly Imipenem and Meropenem [2] . Carbapenemase encoded by OXA23 gene can hydrolyze carbapenem drugs, so that AB carrying this gene is resistant to such drugs which are called the last line of defense against AB . Unfortunately, the incidence of CRAB among clinical isolates from patients with NBSI has been on the rise. According to the China Antimicrobial Surveillance Network (CHINET), the resistance rate of AB to Imipenem and Meropenem in China has increased from 32.9% and 41.3% in 2005 to 68.1% and 69.0% in 2020, and the resistance rate of AB to Imipenem and Meropenem in Belarus and Turkey was 68% and 93% according to the Central Asian and Eastern European surveillance of antimicrobial resistance in 2016 [3–5] . This highlights the urgent need for a reliable and rapid method of differentiation of CSAB and CRAB isolates for optimal treatment. The traditional methods for the detection of CRAB and CSAB are based on blood culture(BC) and include colony morphology and drug sensitivity test [6–7] . However, these methods are time-consuming and laborious, often taking more than three days to identify CRAB. In recent decades, many molecular methods, such as PCR, Real-time quantitative PCR (qPCR)and digital PCR (ddPCR) have been applied for quick detection of pathogenic microorganisms and their resistance genes [8–11] . Hence, we have established a fast and economical PCR-UV method targeting the 16sRNA, OXA23 and OXA51 genes. EXPERIMENTAL DESIGN MATERIALS AND METHODS 1 Materials 1.1 Specimen Source AB strains were isolates form BSIs samples from the First Affiliated Hospital of Guangzhou Medical University. 23 different types of bacteria, fungi, and viruses were collected for specificity test. All strains have been confirmed by mass spectrometry and tested antibiotic susceptibility using the VITEK 2 system. E. coli ATCC25922 and P. aeruginosa (ATCC25923) strains were used as a quality control. The isolation, culture, and identification of specimens were conducted according to the National Clinical Test Operation Procedure [12] . 1.2 Reagents and Instruments The VITEK 2 Compact automatic Microbiology Analyzer and matching Board (Bio-Merieux, France), Drug Sensitive Paper (OXOID), Nucleic Acid Extraction Reagents (Bo’ao Jingdian Biology / Beijing), PCR instrument (BIO-RAD company, USA), NanoDrop 2000 (Thermo Scientific/ USA), electrophoresis instrument (Baijing biology company / Beijing), UV gel imaging and analysis system (Gene Company Limited), ect were used in the study. 2 Experimental Procedure 2.1 DNA Extraction Bacterial DNA was extracted using a column extraction method. [13] . The concentration and purity of the obtained DNA were detected and recorded by the NanoDrop 2000 micro-ultraviolet spectrophotometer. The DNA was stored in the refrigerator at -20℃. The operation process were carried out according to the National Clinical Test Operation Procedure [6] and the Standard of Operation for Clinical Microbial Blood Culture [14] . 3 Establishment and Optimization of Multiple PCR 3.1 Primer Comparison and Optimization Six different sets of 16sRNA primer were combined with OXA23 and OXA51 to form six groups of triple primer combinations. Their amplification product were tested on 2% agarose gel electrophoresis to ensure no dimer between primers, less non-specific amplification, and other conditions. 3.2 Annealing Temperature Optimization This method set a series of gradient annealing temperature for the amplification process, with the aim of determining which temperature has the clearest and most accurate bands. 4. Specificity assessment 23 different microorganisms that are often detected in BSIs were tested under the same conditions. Negative and positive controls were set up. 5. The reproducibility of Multiplex PCR The concentration of bacterial DNA was diluted to a gradient concentration with water. The multiplex PCR experiments were amplified in three replicates for each sample. 6. The UV-Image J Analysis of PCR Products We incorporated Goldview at the beginning of the PCR. The products were directly photographed by imager under ultraviolet light. The corresponding gray values of the images were then quantitatively analyzed by ImageJ. 7. Validation of clinical isolates The DNA of 30 BSIs isolates were tested by PCR-UV assay. The results were compared with PCR electrophoresis and blood culture & drug sensitivity. RESULTS 1. Establishment and Optimization of Multiple PCR 1.1 Primer Comparison and Optimization In Fig. 1 : P(CRAB) should contains three stripes. N1-3(CSAB 1–3) contains two stripes. The blank(B) has no stripes. The results are as below: 1 and 2 sets of 16sRNA primers combination show that both CRAB(P) and CSAB(N1-3) cannot be fully displayed. 4 to 6 sets of 16sRNA primers combination show that CRAB(P) all have three bands but CRAB(N2-3) appear incomplete or non-specific amplification. Only the 3 set of 16sRNA primers combination show correct results. So we choosed the third combination as the best group(Table S1 ). 1.2 Annealing Temperature Optimization The figure illustrates the impact of annealing temperature on the specificity and sensitivity of the PCR amplification for the blaOXA23, blaOXA51, and 16sRNA genes in CRAB. The optimal annealing temperature was determined to be 56.4℃, as it resulted in the strongest and most specific amplification bands on the agarose gel electrophoresis. The sequencing results of blaOXA23 and OXA51 genes were 99% homologous compared to the NCBI sequence(Figure S1 -2). 2. Specificity of Multiplex PCR The amplification results were determined by 2% agarose gel electrophoresis. No specific bands were found in the amplification of 23 different microbial genes in the multiplex PCR system(Fig. 3 ). CRAB and CSAB are the positive control. H 2 O is the blank control. The results showed excellent specificity to AB . 3. The reproducibility and Limit of detection(LOD)of Multiplex PCR Figure 4 DNA Gradient dilution of CRAB DNA showing the detection of OXA23, OXA51, and 16sRNA genes at varying concentrations(ddH2O as the blank control). Amplification of target genes in three replicates demonstrating the stability of the PCR reaction. It was determined that the Limit of detection(LOD) of CRAB was 3.0 x 10 − 1 ng/uL. 4. The UV-Image J Analysis of PCR Products Goldview was added to the PCR system before amplification. Different DNA of the same concentration was added. Amplification result was grayed out by Image J software. The result can be calculated by the gray value of the tube to be tested / the gray value of the blank control of the same batch. The ratio > 1.3 is CRAB, the ratio between 1.1–1.3 is CSAB. the ratio < 1.1 is negative result. At the same time, we also performed electrophoresis analysis on the same tube, and the results were consistent. 5. Validation of clinical isolates The clinical applicability of PCR-UV assay was detected by detecting 30 clinical isolates from BSIs. All results were consistent with blood culture and PCR electrophoresis. Table 1 Clinical Validation of the UV-PCR assay PCR BC Number of bacterial 16sRNA OXA23 OXA51 UV-PCR (ratio) Drug sensitivity results (phenotype) negative 1 - - - < 1.1 - Staphylococcus aureus 2 - - - < 1.1 Carbapenem Sensitive Escherichia coli 2 - - - < 1.1 Carbapenem Sensitive Klebsiella pneumoniae 2 - - - 1.3 Carbapenem Resistance DISCUSSION BSI is a serious global health concern, caused by the invasion of various pathogenic microorganisms into the bloodstream. Bacteria, fungi and viruses are the most common culprits, with bacteria accounting for approximately 90% of all cases [15–18] . Among them, coagulase negative Staphylococcus, AB, Escherichia coli, and Klebsiella pneumoniae are the most frequent offenders [19–22] . The high morbidity and mortality associated with CRAB infections has made the timely diagnosis and identification of CRAB an urgent priority. Traditional diagnostic methods, such as blood culture, Gram staining, colony morphology, biochemical identification, and drug sensitivity tests, are often slow, costly, and inefficient. Molecular diagnostic methods, like ddPCR and real-time PCR, require expensive equipment and reagents. In the face of these challenges, there is an urgent need for a reliable, rapid, cost-effective, and specific assay for the identification of CRAB. So, we devised a multiplex PCR-UV based on the OXA23, OXA51, and 16sRNA. 16sRNA is specific and appears to be uniquely present in AB. Based on our previous study [7] , OXA23 was found to be the predominant carbapenemase genotype of CRAB in BSIs in our hospital. This result is consistent with the majority of domestic and international reports [23–25] . Hence, OXA23 gene is become a powerful predictor for carbapenem resistance [26–31] . Our results show that all CRAB carry OXA23 and OXA51. All CSAB carrying no OXA23. CSAB with OXA51 presents higher resistance rates on Trimethoprim-sulfamethoxazole, Piperacillin-tazobactam and Ciprofloxacin than without. PCR-UV provides time-saving benefits over PCR. Unlike PCR, it does not require electrophoresis to interpret the results. we employed a UV-based and image J software-assisted method. Goldview was added to the PCR system before initiating the PCR reaction, and the products were directly photographed by imager under ultraviolet light. The corresponding gray values of the images were then quantitatively analyzed by ImageJ. Finally, the clinical specimen validation results showed that the results of PCR-UV assay are consistent with those of PCR electrophoresis and blood culture(drug sensitivity) identification. Simultaneously, this method is faster and cheaper than PCR. The entire detection process of PCR-UV assay, including PCR amplification (90 min) and UV-reading and Image J-calculation (approximately 10 min), could be accomplished within 100 min. Moreover, the cost of PCR-UV assay is much lower than that of qPCR and ddPCR. To sum up, PCR-UV assay has shorter detection time and provided a new and more economical detection method for CRAB in BSIs. Limitation First, only CRAB was detected, future research should expand more common microorganisms in BSIs. Secondly, Subsequent clinical validation with large samples and multiple centers is required. CONCLUSION The PCR-UV assay is a useful molecular tool for clinically diagnosis. It has good specificity and sensitivity for the detection of CRAB. The PCR-UV assay is faster and cheaper than PCR. It provides a promising alternative to traditional methods in early diagnosis and treatment of CRAB in BSIs. Declarations RESEARCH FUNDING This research was funded by the Natural Science Foundation of Guangdong Province (2021A1515011057). AUTHORS' CONTRIBUTIONS We indicated the initials for Dr. Xiaodan Zheng and Dr. Xianlin Zhou as XZ1 and XZ2.JZ participated in the primer design and drafted the manuscript. XZ1 participated in the DNA extraction. SL participated in strain identification. YZ participated in antibiotic susceptibility test, XZ2 participated in the amplification of target genes. ZW participated in the design of the study and ZL performed the statistical analysis. SBQ conceived of the study, and participated in its design and coordination. YL helped to draft the manuscript. All authors read and approved the final manuscript. ETHICAL APPROVAL Not applicable. INFORMED CONSENT Not applicable. COMPETING INTERESTS The authors declare that they have no competing interests. The patent substance detected by the UV assay has applied for the China National patent and the patent application has been accepted (Chinese National Invention Patent Application No. 202210770126.1) ACKNOWLEDGEMENT We would like to thank all participants and their families. And thank Dr. Cheng Zhangkai for polishing our article. References Chang, Y.Y., Y.S. Yang, S.L. Wu, et al., Comparison of Cefepime-Cefpirome and Carbapenem Therapy for Acinetobacter Bloodstream Infection in a Multicenter Study. Antimicrob Agents Chemother, 2020. 64(6):33-35. Zhao, Y., K. Hu, J. Zhang, et al., Outbreak of CRAB carrying the carbapenemase OXA23 in ICU of the eastern Heilongjiang Province, China. BMC Infect Dis, 2019. 19(1): 452. Hu, F.P., Y. Guo, D.M. Zhu, et al., Resistance trends among clinical isolates in China reported from CHINET surveillance of bacterial resistance, 2005-2014. Clin Microbiol Infect, 2016. 22 (1): 9-14. Hu Fupin, Guo Yan, Zhu Demei,et al.,CHINET surveillance of bacterial resistance: results of 2020【J】Chinese Journal of Infection and Chemotherapy.2021.21(04):377-387. Redondo González, O., I. Lorenzo Prieto, J. Cobos López, et al., Carbapenamase OXA23 like-producing Acinetobacter baumanii epidemic outbreak in a hospitalization unit. Rev Esp Salud Publica, 2021. 95:21-24. CLSI, C.J.C.L.S.I., Performance standards for antimicrobial susceptibility testing. 2016. 35(3):16-38. Zhang Jingzhi, Yu Lin, Zhang Yandong, et al. Analysis of infection characteristics and drug resistance genes of CRAB in blood culture【J】Chinese Journal of Zoonoses.2022.38(10):874-882 Mavridis,K. , Papapostolou,K.M. , Riga,M. ,,et al."Multiple TaqMan qPCR and droplet digital PCR ( ddPCR ) diagnostics for pesticide resistance monitoring and management, in the major agricultural pest Tetranychus urticae ".Pest Management Science, 2021,78:263-273. Han,Y. , Wang,J. , Zhang,S., et al."Simultaneous quantification of hepatitis A virus and norovirus genogroup I and II by triplex droplet digital PCR".Food Microbiology, 2022, 103:103933. Zhang Z., Wang D., Li Y., et al. Comparison of the Performance of Phenotypic Methods for the Detection of Carbapenem-Resistant Enterobacteriaceae (CRE) in Clinical Practice. Front Cell Infect Microbiol. 2022, 12:849564. Zhou Tingyin,Diagnosis and Illustration of Clinical Microbiology .Shanghai:SHANGHAI SCIENCE&TECHNOLOGY PUBLISHERS. Vanbroekhoven, K., A. Ryngaert, P. Wattiau, et al., Acinetobacter diversity in environmental samples assessed by 16S rRNA gene PCR-DGGE fingerprinting. FEMS Microbiol Ecol, 2004. 50(1): 37-50. Bian, X., X. Liu, X. Zhang, et al., Epidemiological and genomic characteristics of Acinetobacter baumannii from different infection sites using comparative genomics. BMC Genomics, 2021. 22(1): 530. Liu J., Peters BM., Yang L., et al. Antimicrobial Treatment on a Catheter-Related Bloodstream Infection (CRBSI) Case Due to Transition of a Multi-Drug-Resistant Ralstonia mannitolilytica from Commensal to Pathogen during Hospitalization. Antibiotics (Basel). 2022;11(10):1376. Fakih, M.G., A. Bufalino, L. Sturm, et al., Coronavirus disease 2019 (COVID-19) pandemic, central-line-associated bloodstream infection (CLABSI), and catheter-associated urinary tract infection (CAUTI): The urgent need to refocus on hardwiring prevention efforts. Infect Control Hosp Epidemiol, 2022. 43(1): 26-31. Smith, J.T., E.M. Eckhardt, N.B. Hansel, et al., Genomic epidemiology of methicillin-resistant and -susceptible Staphylococcus aureus from bloodstream infections. BMC Infectious Diseases, 2021, 21(1):891-896. Noster, J., M.B. Koeppel, M. Desnos-Olivier, et al., Bloodstream Infections Caused by Magnusiomyces capitatus and Magnusiomyces clavatus: Epidemiological, Clinical, and Microbiological Features of Two Emerging Yeast Species. Antimicrob Agents Chemother, 2022. 66(2): e0183421. Yu, K., W. Zeng, Y. Xu, et al., Bloodstream infections caused by ST2 Acinetobacter baumannii: risk factors, antibiotic regimens, and virulence over 6 years period in China. Antimicrob Resist Infect Control, 2021. 10(1): 16. Dao, T.H., R. Alsallaq, J.B. Parsons, et al., Vancomycin Heteroresistance and Clinical Outcomes in Bloodstream Infections Caused by Coagulase-Negative Staphylococci. Antimicrob Agents Chemother, 2020. 64(11):65-69. Paramita, R.I., E.J. Nelwan, F. Fadilah, et al., Genome-based characterization of Escherichia coli causing bloodstream infection through next-generation sequencing. PLoS One, 2020. 15(12): e0234358. Yang, Y., Y. Yang, G. Chen, et al., Molecular characterization of carbapenem-resistant and virulent plasmids in Klebsiella pneumoniae from patients with bloodstream infections in China. Emerg Microbes Infect, 2021. 10(1):700-709. Garnacho-Montero, J., R. Amaya-Villar, C. Ferrándiz-Millón, et al., Optimum treatment strategies for CRAB bacteremia. Expert Rev Anti Infect Ther, 2015. 13(6): 769-77. Piperaki, E.T., L.S. Tzouvelekis, V. Miriagou, et al., CRAB: in pursuit of an effective treatment. Clin Microbiol Infect, 2019. 25(8): 951-957. Zhang, S., L. Sun, L. Sun, et al., [Application progress of polymyxin in bloodstream infection of drug-resistant Acinetobacter baumannii]. Zhonghua Wei Zhong Bing Ji Jiu Yi Xue, 2021. 33(11): 1401-1404. Abouelfetouh, A., J. Mattock, D. Turner, et al., Diversity of CRAB and bacteriophage-mediated spread of the Oxa23 carbapenemase. Microb Genom, 2022. 8(2):1156-59. Douraghi, M., J.J. Kenyon, P. Aris, et al., Accumulation of Antibiotic Resistance Genes in CRAB Isolates Belonging to Lineage 2, Global Clone 1, from Outbreaks in 2012-2013 at a Tehran Burns Hospital. mSphere, 2020. 5(2):312-318. Nigro, S.J. and R.M. Hall, Structure and context of Acinetobacter transposons carrying the oxa23 carbapenemase gene. J Antimicrob Chemother, 2016. 71(5): 1135-47. Yang, Z., P. Wang, P. Song, et al., Carbapenemase OXA-423: A Novel OXA23 Variant in Acinetobacter baumannii. Infect Drug Resist, 2020, 13:4069-4075. Koirala, J., I. Tyagi, L. Guntupalli, et al., OXA23 and OXA-40 producing CRAB in Central Illinois. Diagn Microbiol Infect Dis, 2020, 97(1):114999. Oliveira, E.A., G.R. Paula, P.J.J. Mondino, et al., High rate of detection of OXA23-producing Acinetobacter from two general hospitals in Brazil. Rev Soc Bras Med Trop, 2019, 52: e20190233. Lombes, A., R.A. Bonnin, F. Laurent, et al., High Prevalence of OXA23 Carbapenemase-Producing Proteus mirabilis among Amoxicillin-Clavulanate-Resistant Isolates in France. Antimicrob Agents Chemother, 2022, 66(2): e0198321. Experimental Design The image depicting the experimental design is available in the Figures section. Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.pdf 124.7.3.pdf Cite Share Download PDF Status: Published Journal Publication published 13 Oct, 2025 Read the published version in Microbial Cell Factories → Version 1 posted Editorial decision: Revision requested 02 Mar, 2025 Reviews received at journal 01 Mar, 2025 Reviews received at journal 24 Feb, 2025 Reviewers agreed at journal 21 Feb, 2025 Reviewers agreed at journal 10 Feb, 2025 Reviewers agreed at journal 15 Nov, 2024 Reviewers agreed at journal 14 Nov, 2024 Reviewers agreed at journal 13 Nov, 2024 Reviewers invited by journal 12 Sep, 2024 Editor assigned by journal 20 Aug, 2024 Submission checks completed at journal 19 Aug, 2024 First submitted to journal 11 Jul, 2024 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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10:27:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4723438/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4723438/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12934-025-02822-w","type":"published","date":"2025-10-13T15:56:54+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":66630596,"identity":"19cbf9c8-6efe-4e75-86b6-c3257d7559a4","added_by":"auto","created_at":"2024-10-15 04:49:35","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":65934,"visible":true,"origin":"","legend":"\u003cp\u003eOptimization of primer groups in CRAB\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4723438/v1/2840998390b04829b3bdd51d.jpg"},{"id":66632255,"identity":"c95fa47f-2f1c-415c-a5eb-6bb4c106e941","added_by":"auto","created_at":"2024-10-15 04:57:35","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":38871,"visible":true,"origin":"","legend":"\u003cp\u003eAmplification results of target genes in CRAB at different annealing temperatures.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4723438/v1/1d63da3942235b5dc3e3a7ce.jpg"},{"id":66630600,"identity":"3f4f800f-b49c-4ea4-9fb7-b2f80da11e41","added_by":"auto","created_at":"2024-10-15 04:49:35","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":67777,"visible":true,"origin":"","legend":"\u003cp\u003eSpecificity of the multiplex PCR.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4723438/v1/a05d6e6d5e59d1cde840111a.jpg"},{"id":66633201,"identity":"f909a5c7-f0a3-4039-9a62-91b415ba9b6a","added_by":"auto","created_at":"2024-10-15 05:05:36","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":20889,"visible":true,"origin":"","legend":"\u003cp\u003eReproducibility of multiplex PCR for the detection of target genes in AB\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4723438/v1/12086d0acdc41caf90c13dd8.jpg"},{"id":66632256,"identity":"b9c53705-f2b1-4e14-80de-e304dc094582","added_by":"auto","created_at":"2024-10-15 04:57:36","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":32084,"visible":true,"origin":"","legend":"\u003cp\u003eUV-Image J\u003cstrong\u003e \u003c/strong\u003eresults of AB-DNA after amplification(Attached electrophoretic comparison results)\u003c/p\u003e\n\u003cp\u003em:Multiple drug resistance CRAB; x:Extensive drug resistance CRAB ; s:CSAB\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4723438/v1/748f284bf0642ed2f41b1dac.jpg"},{"id":66632258,"identity":"736d8764-c535-4ad4-a46c-fe25791c81f5","added_by":"auto","created_at":"2024-10-15 04:57:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":91722,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental Design\u003c/p\u003e","description":"","filename":"Image.png","url":"https://assets-eu.researchsquare.com/files/rs-4723438/v1/e18ea8287c9b39a66f953719.png"},{"id":93955909,"identity":"d49e8a95-c4a1-4e1e-bf1a-25a05df41ca3","added_by":"auto","created_at":"2025-10-20 16:06:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1086443,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4723438/v1/8332c88c-dc19-4cd5-b539-6c2518637c75.pdf"},{"id":66632257,"identity":"520f75ea-9755-45e8-9dda-295f8f1c7b45","added_by":"auto","created_at":"2024-10-15 04:57:36","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":429460,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4723438/v1/3ca233a32d8fd51da3f98717.pdf"},{"id":66630601,"identity":"d5d751d4-a474-45ad-9412-3a53e55bc078","added_by":"auto","created_at":"2024-10-15 04:49:36","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":364585,"visible":true,"origin":"","legend":"","description":"","filename":"124.7.3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4723438/v1/c78f23477badfaa406593606.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development and Clinical Application of a PCR-UV Assay for Detection of Carbapenem Resistant Acinetobacter baumannii in Bloodstream infections ","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003e \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e (AB), a gram-positive bacterium, is a significant human pathogen that has the ability to cause a wide array of severe hospital and community-acquired infections, particularly BSIs\u003csup\u003e[1]\u003c/sup\u003e. BSIs is a major concern in hospitals worldwide, commonly known as nosocomial bloodstream infections (NBSI). \u003cem\u003eAB\u003c/em\u003e is one of the most important opportunistic pathogens causing NBSI due to its natural and acquired resistance to antibiotics, particularly Imipenem and Meropenem \u003csup\u003e[2]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCarbapenemase encoded by OXA23 gene can hydrolyze carbapenem drugs, so that \u003cem\u003eAB\u003c/em\u003e carrying this gene is resistant to such drugs which are called the last line of defense against \u003cem\u003eAB\u003c/em\u003e. Unfortunately, the incidence of CRAB among clinical isolates from patients with NBSI has been on the rise. According to the China Antimicrobial Surveillance Network (CHINET), the resistance rate of AB to Imipenem and Meropenem in China has increased from 32.9% and 41.3% in 2005 to 68.1% and 69.0% in 2020, and the resistance rate of AB to Imipenem and Meropenem in Belarus and Turkey was 68% and 93% according to the Central Asian and Eastern European surveillance of antimicrobial resistance in 2016\u003csup\u003e[3\u0026ndash;5]\u003c/sup\u003e. This highlights the urgent need for a reliable and rapid method of differentiation of CSAB and CRAB isolates for optimal treatment.\u003c/p\u003e \u003cp\u003eThe traditional methods for the detection of CRAB and CSAB are based on blood culture(BC) and include colony morphology and drug sensitivity test \u003csup\u003e[6\u0026ndash;7]\u003c/sup\u003e. However, these methods are time-consuming and laborious, often taking more than three days to identify CRAB. In recent decades, many molecular methods, such as PCR, Real-time quantitative PCR (qPCR)and digital PCR (ddPCR) have been applied for quick detection of pathogenic microorganisms and their resistance genes \u003csup\u003e[8\u0026ndash;11]\u003c/sup\u003e. Hence, we have established a fast and economical PCR-UV method targeting the 16sRNA, OXA23 and OXA51 genes.\u003c/p\u003e"},{"header":"EXPERIMENTAL DESIGN","content":"\u003cp\u003e \u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e1 Materials\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e1.1 Specimen Source\u003c/h2\u003e \u003cp\u003eAB strains were isolates form BSIs samples from the First Affiliated Hospital of Guangzhou Medical University. 23 different types of bacteria, fungi, and viruses were collected for specificity test. All strains have been confirmed by mass spectrometry and tested antibiotic susceptibility using the VITEK 2 system. E. coli ATCC25922 and P. aeruginosa (ATCC25923) strains were used as a quality control. The isolation, culture, and identification of specimens were conducted according to the National Clinical Test Operation Procedure \u003csup\u003e[12]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e1.2 Reagents and Instruments\u003c/h2\u003e \u003cp\u003eThe VITEK 2 Compact automatic Microbiology Analyzer and matching Board (Bio-Merieux, France), Drug Sensitive Paper (OXOID), Nucleic Acid Extraction Reagents (Bo\u0026rsquo;ao Jingdian Biology / Beijing), PCR instrument (BIO-RAD company, USA), NanoDrop 2000 (Thermo Scientific/ USA), electrophoresis instrument (Baijing biology company / Beijing), UV gel imaging and analysis system (Gene Company Limited), ect were used in the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2 Experimental Procedure\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.1 DNA Extraction\u003c/h2\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eBacterial DNA was extracted using a column extraction method.\u003csup\u003e[13]\u003c/sup\u003e. The concentration and purity of the obtained DNA were detected and recorded by the NanoDrop 2000 micro-ultraviolet spectrophotometer. The DNA was stored in the refrigerator at -20℃. The operation process were carried out according to the National Clinical Test Operation Procedure\u003csup\u003e[6]\u003c/sup\u003e and the Standard of Operation for Clinical Microbial Blood Culture \u003csup\u003e[14]\u003c/sup\u003e.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e3 Establishment and Optimization of Multiple PCR\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section4\"\u003e \u003ch2\u003e3.1 Primer Comparison and Optimization\u003c/h2\u003e \u003cp\u003eSix different sets of 16sRNA primer were combined with OXA23 and OXA51 to form six groups of triple primer combinations. Their amplification product were tested on 2% agarose gel electrophoresis to ensure no dimer between primers, less non-specific amplification, and other conditions.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Annealing Temperature Optimization\u003c/h2\u003e \u003cp\u003eThis method set a series of gradient annealing temperature for the amplification process, with the aim of determining which temperature has the clearest and most accurate bands.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4. Specificity assessment\u003c/h2\u003e \u003cp\u003e23 different microorganisms that are often detected in BSIs were tested under the same conditions. Negative and positive controls were set up.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e5. The reproducibility of Multiplex PCR\u003c/h2\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eThe concentration of bacterial DNA was diluted to a gradient concentration with water. The multiplex PCR experiments were amplified in three replicates for each sample.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e6. The UV-Image J Analysis of PCR Products\u003c/h2\u003e \u003cp\u003eWe incorporated Goldview at the beginning of the PCR. The products were directly photographed by imager under ultraviolet light. The corresponding gray values of the images were then quantitatively analyzed by ImageJ.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e7. Validation of clinical isolates\u003c/h2\u003e \u003cp\u003eThe DNA of 30 BSIs isolates were tested by PCR-UV assay. The results were compared with PCR electrophoresis and blood culture \u0026amp; drug sensitivity.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e1. Establishment and Optimization of Multiple PCR\u003c/b\u003e\u003c/h2\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e1.1 Primer Comparison and Optimization\u003c/h2\u003e \u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e: P(CRAB) should contains three stripes. N1-3(CSAB 1\u0026ndash;3) contains two stripes. The blank(B) has no stripes. The results are as below: 1 and 2 sets of 16sRNA primers combination show that both CRAB(P) and CSAB(N1-3) cannot be fully displayed. 4 to 6 sets of 16sRNA primers combination show that CRAB(P) all have three bands but CRAB(N2-3) appear incomplete or non-specific amplification. Only the 3 set of 16sRNA primers combination show correct results. So we choosed the third combination as the best group(Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e1.2 Annealing Temperature Optimization\u003c/h2\u003e \u003cp\u003eThe figure illustrates the impact of annealing temperature on the specificity and sensitivity of the PCR amplification for the blaOXA23, blaOXA51, and 16sRNA genes in CRAB. The optimal annealing temperature was determined to be 56.4℃, as it resulted in the strongest and most specific amplification bands on the agarose gel electrophoresis. The sequencing results of blaOXA23 and OXA51 genes were 99% homologous compared to the NCBI sequence(Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2. Specificity of Multiplex PCR\u003c/h2\u003e \u003cp\u003eThe amplification results were determined by 2% agarose gel electrophoresis. No specific bands were found in the amplification of 23 different microbial genes in the multiplex PCR system(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). CRAB and CSAB are the positive control. H\u003csub\u003e2\u003c/sub\u003eO is the blank control. The results showed excellent specificity to \u003cem\u003eAB\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3. The reproducibility and Limit of detection(LOD)of Multiplex PCR\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e DNA Gradient dilution of CRAB DNA showing the detection of OXA23, OXA51, and 16sRNA genes at varying concentrations(ddH2O as the blank control). Amplification of target genes in three replicates demonstrating the stability of the PCR reaction. It was determined that the Limit of detection(LOD) of CRAB was 3.0 x 10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ng/uL.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e4. The UV-Image J Analysis of PCR Products\u003c/h2\u003e \u003cp\u003eGoldview was added to the PCR system before amplification. Different DNA of the same concentration was added. Amplification result was grayed out by Image J software. The result can be calculated by the gray value of the tube to be tested / the gray value of the blank control of the same batch. The ratio\u0026thinsp;\u0026gt;\u0026thinsp;1.3 is CRAB, the ratio between 1.1\u0026ndash;1.3 is CSAB. the ratio\u0026thinsp;\u0026lt;\u0026thinsp;1.1 is negative result.\u003c/p\u003e \u003cp\u003eAt the same time, we also performed electrophoresis analysis on the same tube, and the results were consistent. \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e5. Validation of clinical isolates\u003c/h2\u003e \u003cp\u003eThe clinical applicability of PCR-UV assay was detected by detecting 30 clinical isolates from BSIs. All results were consistent with blood culture and PCR electrophoresis.\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\u003eClinical Validation of the UV-PCR assay\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" 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 \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;PCR\u003c/p\u003e \u003cp\u003eBC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNumber of bacterial\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16sRNA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOXA23\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eOXA51\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUV-PCR\u003c/p\u003e \u003cp\u003e(ratio)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eDrug sensitivity results\u003c/p\u003e \u003cp\u003e(phenotype)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\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 \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStaphylococcus aureus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\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 \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCarbapenem Sensitive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEscherichia coli\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\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 \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCarbapenem Sensitive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKlebsiella pneumoniae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\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 \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCarbapenem Sensitive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCSAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\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 \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.1\u0026ndash;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCarbapenem Sensitive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCSAB(OXA51)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\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 \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.2\u0026ndash;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCarbapenem Sensitive\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=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\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 \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCarbapenem Resistance\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eBSI is a serious global health concern, caused by the invasion of various pathogenic microorganisms into the bloodstream. Bacteria, fungi and viruses are the most common culprits, with bacteria accounting for approximately 90% of all cases \u003csup\u003e[15\u0026ndash;18]\u003c/sup\u003e. Among them, coagulase negative Staphylococcus, AB, Escherichia coli, and Klebsiella pneumoniae are the most frequent offenders\u003csup\u003e[19\u0026ndash;22]\u003c/sup\u003e. The high morbidity and mortality associated with CRAB infections has made the timely diagnosis and identification of CRAB an urgent priority.\u003c/p\u003e \u003cp\u003eTraditional diagnostic methods, such as blood culture, Gram staining, colony morphology, biochemical identification, and drug sensitivity tests, are often slow, costly, and inefficient. Molecular diagnostic methods, like ddPCR and real-time PCR, require expensive equipment and reagents. In the face of these challenges, there is an urgent need for a reliable, rapid, cost-effective, and specific assay for the identification of CRAB.\u003c/p\u003e \u003cp\u003eSo, we devised a multiplex PCR-UV based on the OXA23, OXA51, and 16sRNA. 16sRNA is specific and appears to be uniquely present in AB. Based on our previous study\u003csup\u003e[7]\u003c/sup\u003e, OXA23 was found to be the predominant carbapenemase genotype of CRAB in BSIs in our hospital. This result is consistent with the majority of domestic and international reports\u003csup\u003e[23\u0026ndash;25]\u003c/sup\u003e. Hence, OXA23 gene is become a powerful predictor for carbapenem resistance\u003csup\u003e[26\u0026ndash;31]\u003c/sup\u003e. Our results show that all CRAB carry OXA23 and OXA51. All CSAB carrying no OXA23. CSAB with OXA51 presents higher resistance rates on Trimethoprim-sulfamethoxazole, Piperacillin-tazobactam and Ciprofloxacin than without.\u003c/p\u003e \u003cp\u003ePCR-UV provides time-saving benefits over PCR. Unlike PCR, it does not require electrophoresis to interpret the results. we employed a UV-based and image J software-assisted method. Goldview was added to the PCR system before initiating the PCR reaction, and the products were directly photographed by imager under ultraviolet light. The corresponding gray values of the images were then quantitatively analyzed by ImageJ.\u003c/p\u003e \u003cp\u003eFinally, the clinical specimen validation results showed that the results of PCR-UV assay are consistent with those of PCR electrophoresis and blood culture(drug sensitivity) identification. Simultaneously, this method is faster and cheaper than PCR. The entire detection process of PCR-UV assay, including PCR amplification (90 min) and UV-reading and Image J-calculation (approximately 10 min), could be accomplished within 100 min. Moreover, the cost of PCR-UV assay is much lower than that of qPCR and ddPCR.\u003c/p\u003e \u003cp\u003eTo sum up, PCR-UV assay has shorter detection time and provided a new and more economical detection method for CRAB in BSIs.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003eLimitation\u003c/h2\u003e \u003cp\u003eFirst, only CRAB was detected, future research should expand more common microorganisms in BSIs. Secondly, Subsequent clinical validation with large samples and multiple centers is required.\u003c/p\u003e \u003c/div\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThe PCR-UV assay is a useful molecular tool for clinically diagnosis. It has good specificity and sensitivity for the detection of CRAB. The PCR-UV assay is faster and cheaper than PCR. It provides a promising alternative to traditional methods in early diagnosis and treatment of CRAB in BSIs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eRESEARCH FUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Natural Science Foundation of Guangdong Province (2021A1515011057).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHORS' CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe indicated the initials for Dr. Xiaodan Zheng and Dr. Xianlin Zhou as XZ1 and XZ2.JZ participated in the primer design and drafted the manuscript. XZ1 participated in the DNA extraction. SL participated in strain identification. YZ participated in antibiotic susceptibility test, XZ2 participated in the amplification of target genes. ZW participated in the design of the study and ZL performed the statistical analysis. SBQ conceived of the study, and participated in its design and coordination. YL helped to draft the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICAL APPROVAL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eINFORMED CONSENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\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\n\u003cp\u003eThe patent substance detected by the UV assay has applied for the China National patent and the patent application has been accepted (Chinese National Invention Patent Application No. 202210770126.1)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank all participants and their families. And thank Dr. Cheng Zhangkai for polishing our article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChang, Y.Y., Y.S. Yang, S.L. Wu, et al., Comparison of Cefepime-Cefpirome and Carbapenem Therapy for Acinetobacter Bloodstream Infection in a Multicenter Study. Antimicrob Agents Chemother, 2020. 64(6):33-35.\u003c/li\u003e\n\u003cli\u003eZhao, Y., K. Hu, J. Zhang, et al., Outbreak of CRAB carrying the carbapenemase OXA23 in ICU of the eastern Heilongjiang Province, China. BMC Infect Dis, 2019. 19(1): 452.\u003c/li\u003e\n\u003cli\u003eHu, F.P., Y. Guo, D.M. Zhu, et al., Resistance trends among clinical isolates in China reported from CHINET surveillance of bacterial resistance, 2005-2014. Clin Microbiol Infect, 2016. 22 (1): 9-14.\u003c/li\u003e\n\u003cli\u003eHu Fupin, Guo Yan, Zhu Demei,et al.,CHINET surveillance of bacterial resistance: results of 2020【J】Chinese Journal of Infection and Chemotherapy.2021.21(04):377-387.\u003c/li\u003e\n\u003cli\u003eRedondo Gonz\u0026aacute;lez, O., I. Lorenzo Prieto, J. Cobos L\u0026oacute;pez, et al., Carbapenamase OXA23 like-producing Acinetobacter baumanii epidemic outbreak in a hospitalization unit. Rev Esp Salud Publica, 2021. 95:21-24.\u003c/li\u003e\n\u003cli\u003eCLSI, C.J.C.L.S.I., Performance standards for antimicrobial susceptibility testing. 2016. 35(3):16-38.\u003c/li\u003e\n\u003cli\u003eZhang Jingzhi, Yu Lin, Zhang Yandong, et al. Analysis of infection characteristics and drug resistance genes of CRAB in blood culture【J】Chinese Journal of Zoonoses.2022.38(10):874-882\u003c/li\u003e\n\u003cli\u003eMavridis,K. , Papapostolou,K.M. , Riga,M. ,,et al.\u0026quot;Multiple TaqMan qPCR and droplet digital PCR ( ddPCR ) diagnostics for pesticide resistance monitoring and management, in the major agricultural pest Tetranychus urticae \u0026quot;.Pest Management Science, 2021,78:263-273.\u003c/li\u003e\n\u003cli\u003eHan,Y. , Wang,J. , Zhang,S., et al.\u0026quot;Simultaneous quantification of hepatitis A virus and norovirus genogroup I and II by triplex droplet digital PCR\u0026quot;.Food Microbiology, 2022, 103:103933.\u003c/li\u003e\n\u003cli\u003eZhang Z., Wang D., Li Y., et al. Comparison of the Performance of Phenotypic Methods for the Detection of Carbapenem-Resistant Enterobacteriaceae (CRE) in Clinical Practice. Front Cell Infect Microbiol. 2022, 12:849564. \u003c/li\u003e\n\u003cli\u003eZhou Tingyin,Diagnosis and Illustration of Clinical Microbiology .Shanghai:SHANGHAI SCIENCE\u0026amp;TECHNOLOGY PUBLISHERS.\u003c/li\u003e\n\u003cli\u003eVanbroekhoven, K., A. Ryngaert, P. Wattiau, et al., Acinetobacter diversity in environmental samples assessed by 16S rRNA gene PCR-DGGE fingerprinting. FEMS Microbiol Ecol, 2004. 50(1): 37-50. \u003c/li\u003e\n\u003cli\u003eBian, X., X. Liu, X. Zhang, et al., Epidemiological and genomic characteristics of Acinetobacter baumannii from different infection sites using comparative genomics. BMC Genomics, 2021. 22(1): 530.\u003c/li\u003e\n\u003cli\u003eLiu J., Peters BM., Yang L., et al. Antimicrobial Treatment on a Catheter-Related Bloodstream Infection (CRBSI) Case Due to Transition of a Multi-Drug-Resistant Ralstonia mannitolilytica from Commensal to Pathogen during Hospitalization. Antibiotics (Basel). 2022;11(10):1376. \u003c/li\u003e\n\u003cli\u003eFakih, M.G., A. Bufalino, L. Sturm, et al., Coronavirus disease 2019 (COVID-19) pandemic, central-line-associated bloodstream infection (CLABSI), and catheter-associated urinary tract infection (CAUTI): The urgent need to refocus on hardwiring prevention efforts. Infect Control Hosp Epidemiol, 2022. 43(1): 26-31.\u003c/li\u003e\n\u003cli\u003eSmith, J.T., E.M. Eckhardt, N.B. Hansel, et al., Genomic epidemiology of methicillin-resistant and -susceptible Staphylococcus aureus from bloodstream infections. BMC Infectious Diseases, 2021, 21(1):891-896.\u003c/li\u003e\n\u003cli\u003eNoster, J., M.B. Koeppel, M. Desnos-Olivier, et al., Bloodstream Infections Caused by Magnusiomyces capitatus and Magnusiomyces clavatus: Epidemiological, Clinical, and Microbiological Features of Two Emerging Yeast Species. Antimicrob Agents Chemother, 2022. 66(2): e0183421.\u003c/li\u003e\n\u003cli\u003eYu, K., W. Zeng, Y. Xu, et al., Bloodstream infections caused by ST2 Acinetobacter baumannii: risk factors, antibiotic regimens, and virulence over 6 years period in China. Antimicrob Resist Infect Control, 2021. 10(1): 16.\u003c/li\u003e\n\u003cli\u003eDao, T.H., R. Alsallaq, J.B. Parsons, et al., Vancomycin Heteroresistance and Clinical Outcomes in Bloodstream Infections Caused by Coagulase-Negative Staphylococci. Antimicrob Agents Chemother, 2020. 64(11):65-69.\u003c/li\u003e\n\u003cli\u003eParamita, R.I., E.J. Nelwan, F. Fadilah, et al., Genome-based characterization of Escherichia coli causing bloodstream infection through next-generation sequencing. PLoS One, 2020. 15(12): e0234358.\u003c/li\u003e\n\u003cli\u003eYang, Y., Y. Yang, G. Chen, et al., Molecular characterization of carbapenem-resistant and virulent plasmids in Klebsiella pneumoniae from patients with bloodstream infections in China. Emerg Microbes Infect, 2021. 10(1):700-709.\u003c/li\u003e\n\u003cli\u003eGarnacho-Montero, J., R. Amaya-Villar, C. Ferr\u0026aacute;ndiz-Mill\u0026oacute;n, et al., Optimum treatment strategies for CRAB bacteremia. Expert Rev Anti Infect Ther, 2015. 13(6): 769-77.\u003c/li\u003e\n\u003cli\u003ePiperaki, E.T., L.S. Tzouvelekis, V. Miriagou, et al., CRAB: in pursuit of an effective treatment. Clin Microbiol Infect, 2019. 25(8): 951-957.\u003c/li\u003e\n\u003cli\u003eZhang, S., L. Sun, L. Sun, et al., [Application progress of polymyxin in bloodstream infection of drug-resistant Acinetobacter baumannii]. Zhonghua Wei Zhong Bing Ji Jiu Yi Xue, 2021. 33(11): 1401-1404.\u003c/li\u003e\n\u003cli\u003eAbouelfetouh, A., J. Mattock, D. Turner, et al., Diversity of CRAB and bacteriophage-mediated spread of the Oxa23 carbapenemase. Microb Genom, 2022. 8(2):1156-59.\u003c/li\u003e\n\u003cli\u003eDouraghi, M., J.J. Kenyon, P. Aris, et al., Accumulation of Antibiotic Resistance Genes in CRAB Isolates Belonging to Lineage 2, Global Clone 1, from Outbreaks in 2012-2013 at a Tehran Burns Hospital. mSphere, 2020. 5(2):312-318.\u003c/li\u003e\n\u003cli\u003eNigro, S.J. and R.M. Hall, Structure and context of Acinetobacter transposons carrying the oxa23 carbapenemase gene. J Antimicrob Chemother, 2016. 71(5): 1135-47.\u003c/li\u003e\n\u003cli\u003eYang, Z., P. Wang, P. Song, et al., Carbapenemase OXA-423: A Novel OXA23 Variant in Acinetobacter baumannii. Infect Drug Resist, 2020, 13:4069-4075.\u003c/li\u003e\n\u003cli\u003eKoirala, J., I. Tyagi, L. Guntupalli, et al., OXA23 and OXA-40 producing CRAB in Central Illinois. Diagn Microbiol Infect Dis, 2020, 97(1):114999.\u003c/li\u003e\n\u003cli\u003eOliveira, E.A., G.R. Paula, P.J.J. Mondino, et al., High rate of detection of OXA23-producing Acinetobacter from two general hospitals in Brazil. Rev Soc Bras Med Trop, 2019, 52: e20190233.\u003c/li\u003e\n\u003cli\u003eLombes, A., R.A. Bonnin, F. Laurent, et al., High Prevalence of OXA23 Carbapenemase-Producing Proteus mirabilis among Amoxicillin-Clavulanate-Resistant Isolates in France. Antimicrob Agents Chemother, 2022, 66(2): e0198321.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Experimental Design","content":"\u003cp\u003eThe image depicting the experimental design is available in the Figures section.\u003c/p\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":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"carbapenem-resistant Acinetobacter baumannii(CRAB), carbapenem-resistance gene, Bloodstream infections(BSIs), PCR-UV assay","lastPublishedDoi":"10.21203/rs.3.rs-4723438/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4723438/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003e Bloodstream infections caused by carbapenem-resistant Acinetobacter baumannii (CRAB) are a significant public health concern, with high morbidity and mortality rates. The detection and identification of CRAB is essential for early diagnosis and treatment. Hence, a rapid and economical CRAB-associated Bloodstream infections(BSIs) detection method is of urgent need.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eA tripling PCR-UV reaction system was designed\u003c/p\u003e\n\u003cp\u003efor detecting the antibiotic resistance gene OXA23, OXA51 and AB-specific gene. The specificity of the primers , limit of detection (LOD), reproducibility, and accuracy of the assay were evaluated. The PCR products were analyzed using UV and ImageJ analysis directly, which provided a quickly interpretation of the results. Furthermore, the established assay was validated on clinical isolates and compared with blood culture and drug sensitivity tests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The tripling PCR-UV method established in this study demonstrated strong primer specificity, distinguishing CRAB among 23 common clinical pathogens. The results of this PCR method have been validated by electrophoresis for good accuracy and reproducibility, with a Limit of Detection (LOD) of 3.0 x 10\u003csup\u003e-1\u003c/sup\u003e ng/uL. Meanwhile, the optimal annealing temperature for the triple method was optimized to 56.4 ℃. The result of PCR amplification could be judged by the result of the gray value of the tube to be tested / the gray value of the blank control of the same batch. The ratio\u0026gt;1.3 is CRAB, the ratio between 1.1-1.3 is carbapenem-sensitive Acinetobacter baumannii (CSAB), the ratio\u0026lt;1.1 is negative result. When applied to detect 30 patients with BSIs of AB, the results were consistent with clinical blood culture identification and drug sensitivity tests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eThe tripling PCR-UV assay developed in this study is a UV-visual, rapid, and cost-effective method for the detection of \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e(AB\u003cem\u003e)\u003c/em\u003e and identification of CRAB in bloodstream infections. The assay could be particularly useful in grassroots units where expensive molecular instruments are not readily available and could help in the diagnosis and treatment of CRAB infections in BSIs.\u003c/p\u003e","manuscriptTitle":"Development and Clinical Application of a PCR-UV Assay for Detection of Carbapenem Resistant Acinetobacter baumannii in Bloodstream infections ","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-15 04:49:31","doi":"10.21203/rs.3.rs-4723438/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-03-02T20:15:01+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-02T00:31:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-02-24T09:13:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"35768919942405705589450812770156579050","date":"2025-02-21T10:10:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"259128313101078858473754509079798532494","date":"2025-02-10T13:13:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"202862937128460996331927741764877847340","date":"2024-11-15T16:35:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"270518013059950094691181756647634341182","date":"2024-11-14T18:54:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"86304565184259672909782898646692598560","date":"2024-11-13T16:41:15+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-12T22:13:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-20T19:18:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-20T03:22:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microbial Cell Factories","date":"2024-07-11T10:25:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6a17ce34-d609-4e88-b0f1-e0e5e2a4e982","owner":[],"postedDate":"October 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-20T15:59:32+00:00","versionOfRecord":{"articleIdentity":"rs-4723438","link":"https://doi.org/10.1186/s12934-025-02822-w","journal":{"identity":"microbial-cell-factories","isVorOnly":false,"title":"Microbial Cell Factories"},"publishedOn":"2025-10-13 15:56:54","publishedOnDateReadable":"October 13th, 2025"},"versionCreatedAt":"2024-10-15 04:49:31","video":"","vorDoi":"10.1186/s12934-025-02822-w","vorDoiUrl":"https://doi.org/10.1186/s12934-025-02822-w","workflowStages":[]},"version":"v1","identity":"rs-4723438","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4723438","identity":"rs-4723438","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}