{"title":"重组酶辅助扩增测定法用于快速检测耐亚胺培南铜绿假单胞菌和耐利福平铜绿假单胞菌","authors":"Yao Zhou, Ruiqing Shi, Liang Mu, Linlin Tian, Mengshan Zhou, Wenhan Lyu, Yaodong Chen","doi":"10.3389/fcimb.2024.1428827","DOIUrl":null,"url":null,"abstract":"The indiscriminate use of antibiotics has resulted in a growing resistance to drugs in <jats:italic>Pseudomonas aeruginosa</jats:italic>. The identification of antibiotic resistance genes holds considerable clinical significance for prompt diagnosis. In this study, we established and optimized a Recombinase-Aided Amplification (RAA) assay to detect two genes associated with drug resistance, <jats:italic>oprD</jats:italic> and <jats:italic>arr</jats:italic>, in 101 clinically collected <jats:italic>P. aeruginosa</jats:italic> isolates. Through screening for the detection or absence of <jats:italic>oprD</jats:italic> and <jats:italic>arr</jats:italic>, the results showed that there were 52 Imipenem-resistant <jats:italic>P. aeruginosa</jats:italic> (IRPA) strains and 23 Rifampin-resistant <jats:italic>P. aeruginosa</jats:italic> (RRPA) strains. This method demonstrated excellent detection performance even when the sample concentration is 10 copies/μL at isothermal conditions and the results could be obtained within 20 minutes. The detection results were in accordance with the results of conventional PCR and Real-time PCR. The detection outcomes of the <jats:italic>arr</jats:italic> gene were consistently with the resistance spectrum. However, the antimicrobial susceptibility results revealed that 65 strains were resistant to imipenem, while 49 strains sensitive to imipenem with <jats:italic>oprD</jats:italic> were identified. This discrepancy could be attributed to genetic mutations. In summary, the RAA has higher sensitivity, shorter time, and lower-cost instrument requirements than traditional detection methods. In addition, to analyze the epidemiological characteristics of the aforementioned drug-resistant strains, we conducted Multilocus Sequence Typing (MLST), virulence gene, and antimicrobial susceptibility testing. MLST analysis showed a strong correlation between the sequence types ST-1639, ST-639, ST-184 and IRPA, while ST-261 was the main subtype of RRPA. It was observed that these drug-resistant strains all possess five or more virulence genes, among which <jats:italic>exoS</jats:italic> and <jats:italic>exoU</jats:italic> do not coexist, and they are all multidrug-resistant strains. The non-coexistence of <jats:italic>exoU</jats:italic> and <jats:italic>exoS</jats:italic> in <jats:italic>P.aeruginosa</jats:italic> is related to various factors including bacterial regulatory mechanisms and pathogenic mechanisms. This indicates that the relationship between the presence of virulence genes and the severity of patient infection is worthy of attention. In conclusion, we have developed a rapid and efficient RAA (Recombinase-Aided Amplification) detection method that offers significant advantages in terms of speed, simplicity, and cost-effectiveness (especially in time and equipment aspect). This novel approach is designed to meet the demands of clinical diagnostics.","PeriodicalId":12458,"journal":{"name":"Frontiers in Cellular and Infection Microbiology","volume":null,"pages":null},"PeriodicalIF":4.6000,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Recombinase-aided amplification assay for rapid detection of imipenem-resistant Pseudomonas aeruginosa and rifampin-resistant Pseudomonas aeruginosa\",\"authors\":\"Yao Zhou, Ruiqing Shi, Liang Mu, Linlin Tian, Mengshan Zhou, Wenhan Lyu, Yaodong Chen\",\"doi\":\"10.3389/fcimb.2024.1428827\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"The indiscriminate use of antibiotics has resulted in a growing resistance to drugs in <jats:italic>Pseudomonas aeruginosa</jats:italic>. The identification of antibiotic resistance genes holds considerable clinical significance for prompt diagnosis. In this study, we established and optimized a Recombinase-Aided Amplification (RAA) assay to detect two genes associated with drug resistance, <jats:italic>oprD</jats:italic> and <jats:italic>arr</jats:italic>, in 101 clinically collected <jats:italic>P. aeruginosa</jats:italic> isolates. Through screening for the detection or absence of <jats:italic>oprD</jats:italic> and <jats:italic>arr</jats:italic>, the results showed that there were 52 Imipenem-resistant <jats:italic>P. aeruginosa</jats:italic> (IRPA) strains and 23 Rifampin-resistant <jats:italic>P. aeruginosa</jats:italic> (RRPA) strains. This method demonstrated excellent detection performance even when the sample concentration is 10 copies/μL at isothermal conditions and the results could be obtained within 20 minutes. The detection results were in accordance with the results of conventional PCR and Real-time PCR. The detection outcomes of the <jats:italic>arr</jats:italic> gene were consistently with the resistance spectrum. However, the antimicrobial susceptibility results revealed that 65 strains were resistant to imipenem, while 49 strains sensitive to imipenem with <jats:italic>oprD</jats:italic> were identified. This discrepancy could be attributed to genetic mutations. In summary, the RAA has higher sensitivity, shorter time, and lower-cost instrument requirements than traditional detection methods. In addition, to analyze the epidemiological characteristics of the aforementioned drug-resistant strains, we conducted Multilocus Sequence Typing (MLST), virulence gene, and antimicrobial susceptibility testing. MLST analysis showed a strong correlation between the sequence types ST-1639, ST-639, ST-184 and IRPA, while ST-261 was the main subtype of RRPA. It was observed that these drug-resistant strains all possess five or more virulence genes, among which <jats:italic>exoS</jats:italic> and <jats:italic>exoU</jats:italic> do not coexist, and they are all multidrug-resistant strains. The non-coexistence of <jats:italic>exoU</jats:italic> and <jats:italic>exoS</jats:italic> in <jats:italic>P.aeruginosa</jats:italic> is related to various factors including bacterial regulatory mechanisms and pathogenic mechanisms. This indicates that the relationship between the presence of virulence genes and the severity of patient infection is worthy of attention. In conclusion, we have developed a rapid and efficient RAA (Recombinase-Aided Amplification) detection method that offers significant advantages in terms of speed, simplicity, and cost-effectiveness (especially in time and equipment aspect). This novel approach is designed to meet the demands of clinical diagnostics.\",\"PeriodicalId\":12458,\"journal\":{\"name\":\"Frontiers in Cellular and Infection Microbiology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":4.6000,\"publicationDate\":\"2024-09-10\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Frontiers in Cellular and Infection Microbiology\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.3389/fcimb.2024.1428827\",\"RegionNum\":2,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"IMMUNOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Frontiers in Cellular and Infection Microbiology","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.3389/fcimb.2024.1428827","RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"IMMUNOLOGY","Score":null,"Total":0}
Recombinase-aided amplification assay for rapid detection of imipenem-resistant Pseudomonas aeruginosa and rifampin-resistant Pseudomonas aeruginosa
The indiscriminate use of antibiotics has resulted in a growing resistance to drugs in Pseudomonas aeruginosa. The identification of antibiotic resistance genes holds considerable clinical significance for prompt diagnosis. In this study, we established and optimized a Recombinase-Aided Amplification (RAA) assay to detect two genes associated with drug resistance, oprD and arr, in 101 clinically collected P. aeruginosa isolates. Through screening for the detection or absence of oprD and arr, the results showed that there were 52 Imipenem-resistant P. aeruginosa (IRPA) strains and 23 Rifampin-resistant P. aeruginosa (RRPA) strains. This method demonstrated excellent detection performance even when the sample concentration is 10 copies/μL at isothermal conditions and the results could be obtained within 20 minutes. The detection results were in accordance with the results of conventional PCR and Real-time PCR. The detection outcomes of the arr gene were consistently with the resistance spectrum. However, the antimicrobial susceptibility results revealed that 65 strains were resistant to imipenem, while 49 strains sensitive to imipenem with oprD were identified. This discrepancy could be attributed to genetic mutations. In summary, the RAA has higher sensitivity, shorter time, and lower-cost instrument requirements than traditional detection methods. In addition, to analyze the epidemiological characteristics of the aforementioned drug-resistant strains, we conducted Multilocus Sequence Typing (MLST), virulence gene, and antimicrobial susceptibility testing. MLST analysis showed a strong correlation between the sequence types ST-1639, ST-639, ST-184 and IRPA, while ST-261 was the main subtype of RRPA. It was observed that these drug-resistant strains all possess five or more virulence genes, among which exoS and exoU do not coexist, and they are all multidrug-resistant strains. The non-coexistence of exoU and exoS in P.aeruginosa is related to various factors including bacterial regulatory mechanisms and pathogenic mechanisms. This indicates that the relationship between the presence of virulence genes and the severity of patient infection is worthy of attention. In conclusion, we have developed a rapid and efficient RAA (Recombinase-Aided Amplification) detection method that offers significant advantages in terms of speed, simplicity, and cost-effectiveness (especially in time and equipment aspect). This novel approach is designed to meet the demands of clinical diagnostics.
期刊介绍:
Frontiers in Cellular and Infection Microbiology is a leading specialty journal, publishing rigorously peer-reviewed research across all pathogenic microorganisms and their interaction with their hosts. Chief Editor Yousef Abu Kwaik, University of Louisville is supported by an outstanding Editorial Board of international experts. This multidisciplinary open-access journal is at the forefront of disseminating and communicating scientific knowledge and impactful discoveries to researchers, academics, clinicians and the public worldwide.
Frontiers in Cellular and Infection Microbiology includes research on bacteria, fungi, parasites, viruses, endosymbionts, prions and all microbial pathogens as well as the microbiota and its effect on health and disease in various hosts. The research approaches include molecular microbiology, cellular microbiology, gene regulation, proteomics, signal transduction, pathogenic evolution, genomics, structural biology, and virulence factors as well as model hosts. Areas of research to counteract infectious agents by the host include the host innate and adaptive immune responses as well as metabolic restrictions to various pathogenic microorganisms, vaccine design and development against various pathogenic microorganisms, and the mechanisms of antibiotic resistance and its countermeasures.