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Molecular detection of bacterial contamination in plasma using magnetic-based enrichment.

ABSTRACT: Bacterial contamination of blood products is a major problem in transfusion medicine, in terms of both morbidity and mortality. Platelets (PLTs) are stored at room temperature (under constant agitation) for more than 5 days, and bacteria can thus grow significantly from a low level to high titers. However, conventional methods like blood culture and lateral flow assay have disadvantages such as long detection time, low sensitivity, and the need for a large volume of blood components. We used real-time polymerase chain reaction (PCR) assays with antibiotic-conjugated magnetic nanobeads (MNBs) to detect enriched Gram-positive and -negative bacteria. The MNBs were coated with polyethylene glycol (PEG) to prevent aggregation by blood components. Over 80% of all bacteria were captured by the MNBs, and the levels of detection were 10¹ colony forming unit [CFU]/mL and 10² CFU/mL for Gram-positive and -negative bacteria, respectively. The detection time is < 3 h using only small volumes of blood components. Thus, compared to conventional methods, real-time PCR using MNBs allows for rapid detection with high sensitivity using only a small volume of blood components.

SUBMITTER: Lee J

PROVIDER: S-EPMC9160056 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Molecular detection of bacterial contamination in plasma using magnetic-based enrichment.

Lee Jinyeop J Abafogi Abdurhaman Teyib AT Oh Sujin S Chang Ho Eun HE Tepeng Wu W Lee Daekyu D Park Sungsu S Park Kyoung Un KU Hong Yun Ji YJ

Scientific reports 20220601 1

Bacterial contamination of blood products is a major problem in transfusion medicine, in terms of both morbidity and mortality. Platelets (PLTs) are stored at room temperature (under constant agitation) for more than 5 days, and bacteria can thus grow significantly from a low level to high titers. However, conventional methods like blood culture and lateral flow assay have disadvantages such as long detection time, low sensitivity, and the need for a large volume of blood components. We used rea ...[more]

PMID: 35650226

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Project description:Bacterial contamination of blood products poses a significant risk in transfusion medicine. Platelets are particularly vulnerable to bacterial growth because they must be stored at room temperature with constant agitation for >5 days. The limitations of bacterial detection using conventional methods, such as blood cultures and lateral flow assays, include the long detection times, low sensitivity, and the requirement for substantial volumes of blood components. To address these limitations, we assessed the performance of a bacterial enrichment technique using antibiotic-conjugated magnetic nanobeads (AcMNBs) and real-time PCR for the detection of bacterial contamination in plasma. AcMNBs successfully captured >80% of four bacterial strains, including Staphylococcus aureus, Bacillus cereus, Escherichia coli, and Klebsiella pneumoniae, in both plasma and phosphate-buffered saline. After 24-h incubation with bacterial enrichment, S. aureus and B. cereus were each detected at 101 CFU/mL in all trials (5/5), E. coli at 101 CFU/mL in 1/5 trials, and K. pneumoniae at 10² CFU/mL in 4/5 trials. Additionally, without incubation, the improvement was also achieved in samples with bacterial enrichment, S. aureus at 10² CFU/mL and B. cereus at 101 CFU/mL in 1/5 trials each, E. coli at 10³ CFU/mL in 3/5 trials, and K. pneumoniae at 10¹ CFU/mL in 2/5 trials. Overall, the findings from this study strongly support the superiority of bacterial enrichment in detecting low-level bacterial contamination in plasma when employing AcMNBs and PCR.IMPORTANCEThe study presents a breakthrough approach to detect bacterial contamination in plasma, a critical concern in transfusion medicine. Traditional methods, such as blood cultures and lateral flow assays, are hampered by slow detection times, low sensitivity, and the need for large blood sample volumes. Our research introduces a novel technique using antibiotic-conjugated magnetic nanobeads combined with real-time PCR, enhancing the detection of bacteria in blood products, especially platelets. This method has shown exceptional efficiency in identifying even low levels of four different species of bacteria in plasma. The ability to detect bacterial contamination rapidly and accurately is vital for ensuring the safety of blood transfusions and can significantly reduce the risk of infections transmitted through blood products. This advancement is a pivotal step in improving patient outcomes and elevating the standards of care in transfusion medicine.

Project description:Modern isothermal microcalorimeters (IMC) are able to detect the metabolic heat of bacteria with an accuracy sufficient to recognize even the smallest traces of bacterial contamination of water, food, and medical samples. The modern IMC techniques are often superior to conventional detection methods in terms of the detection time, reliability, labor, and technical effort. What is missing is a systematic analysis of the influence of the cultivation conditions on calorimetric detection. For the acceptance of IMC techniques, it is advantageous if already standardized cultivation techniques can be combined with calorimetry. Here we performed such a systematic analysis using Lactobacillus plantarum as a model bacterium. Independent of the cultivation techniques, IMC detections were much faster for high bacterial concentrations (>102 CFU?mL-1) than visual detections. At low bacterial concentrations (<102 CFU?mL-1), detection times were approximately the same. Our data demonstrate that all kinds of traditional cultivation techniques like growth on agar (GOA) or in agar (GIA), in liquid media (GL) or on agar after enrichment via membrane filtration (GF) can be combined with IMC. The order of the detection times was GF < GIA ? GL ? GOA. The observed linear relationship between the calorimetric detection times and the initial bacterial concentrations can be used to quantify the bacterial contamination. Further investigations regarding the correlation between the filling level (in mm) of the calorimetric vessel and the specific maximum heat flow (in ?W?g-1) illustrated two completely different results for liquid and solid media. Due to the better availability of substrates and the homogeneous distribution of bacteria growing in a liquid medium, the volume-related maximum heat flow was independent on the filling level of the calorimetric vessels. However, in a solid medium, the volume-related maximum heat flow approached a threshold and achieved a maximum at low filling levels. This fundamentally different behavior can be explained by the spatial limitation of the growth of bacterial colonies and the reduced substrate supply due to diffusion.

Dataset Information

Molecular detection of bacterial contamination in plasma using magnetic-based enrichment.

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Molecular detection of bacterial contamination in plasma using magnetic-based enrichment.

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