Same-day confirmation of infection and antimicrobial susceptibility profiling using flow cytometry

Mulroney, Kieran T.; Kopczyk, Margaret; Carson, Christine; Paton, Teagan; Inglis, Timothy J. J.; Chakera, Aron

doi:10.1016/j.ebiom.2022.104145

Cited by 9 publications

(12 citation statements)

References 23 publications

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“…Therefore, the sensitivity of this flocculation-based technique is well below this threshold. In a clinical setting, rapid diagnostic tests are often used alongside other methods that confirm the presence of an infection . When combined with slower, established methods such as bacterial culture to confirm a true positive result, this gives confidence in a positive result and benefits patients within the clinical setting by providing a quicker time to result for commencement or cessation of antimicrobial treatment.…”

Section: Resultsmentioning

confidence: 99%

Bridging Flocculation of a Sterically Stabilized Cationic Latex as a Biosensor for the Detection of Microbial DNA after Amplification via PCR

Trinh,

Batt,

Yue

et al. 2024

Biomacromolecules

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There is a high demand for rapid, sensitive, and accurate detection methods for pathogens. This paper demonstrates a method of detecting the presence of amplified DNA from a range of pathogens associated with serious infections including Gram-negative bacteria, Gram-positive bacteria, and viruses. DNA is amplified using a polymerase chain reaction (PCR) and consequently detected using a sterically stabilized, cationic polymer latex. The DNA induces flocculation of this cationic latex, which consequently leads to rapid sedimentation and a visible change from a milky-white dispersion to one with a transparent supernatant, presenting a clear visible change, indicating the presence of amplified DNA. Specifically, a number of different pathogens were amplified using conventional or qPCR, including Staphylococcus aureus, Escherichia coli, and Herpes Simplex Virus (HSV-2). This method was demonstrated to detect the presence of bacteria in suspension concentrations greater than 380 CFU mL −1 and diagnose the presence of specific genomes through primer selection, as exemplified using methicillin resistant and methicillin susceptible Staphylococcus aureus. The versatility of this methodology was further demonstrated by showing that false positive results do not occur when a PCR of fungal DNA from C. albicans is conducted using bacterial universal primers.

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Section: Resultsmentioning

confidence: 99%

Bridging Flocculation of a Sterically Stabilized Cationic Latex as a Biosensor for the Detection of Microbial DNA after Amplification via PCR

Trinh,

Batt,

Yue

et al. 2024

Biomacromolecules

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show abstract

“…However, they take several days to provide a result because they require the isolation of bacteria isolated from patients who were then exposed to the antibiotics. To overcome this delay in conventional AST assays, new phenotypic strategies have been proposed for the rapid measurement of various phenotypic characteristics of bacteria, such as morphology, metabolism, biochemical composition, and growth after exposure to antibiotics, including isothermal microcalorimetry [ 28 , 29 ], electrochemical impedance spectroscopy [ 30 , 31 ], microscopy [ 32 , 33 ], electrochemical ASTs [ 34 , 35 ], spectroscopy [ 36 ], flow cytometry [ 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 ], and spectrometry [ 46 ]. Rapid phenotypic AST methods have detected microbial growth and/or metabolism as well as morphological changes in biological samples with very low microbial loads.…”

Section: The Landscape Of Rapid Methods For Antibiotic Susceptibility...mentioning

confidence: 99%

“…Table 1 provides examples of FCM-based AST approaches developed for the detection of AMR in pathogenic bacteria. FCM assays were demonstrated to accurately and rapidly detect AMR profiles of pathogens directly in clinical samples [ 37 , 38 , 42 , 47 , 48 , 49 , 50 , 51 , 52 , 53 ]. Most of the developed AST methods based on FCM used fluorescent dyes to assess the viability of microbial cells after exposure to antibiotics.…”

Section: Rapid Detection Of Bacterial Pathogens and Resistance–contri...mentioning

confidence: 99%

“…However, the progress of phenotypic ASTs based on FCM assays has been hindered by the different interactions between bacteria and antibiotics, the nonspecific binding of fluorescent dyes, and the limited computational power to detect changes within heterogeneous populations [ 18 ]. However, recently, commercial ASTs based on FCM have been developed to provide AST reports within 2 h, instead of 24–48 h in the case of current standard methods [ 39 , 40 , 41 , 42 ].…”

Section: Rapid Detection Of Bacterial Pathogens and Resistance–contri...mentioning

confidence: 99%

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Current and Future Flow Cytometry Applications Contributing to Antimicrobial Resistance Control

Măruţescu

2023

Microorganisms

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Antimicrobial resistance is a global threat to human health and welfare, food safety, and environmental health. The rapid detection and quantification of antimicrobial resistance are important for both infectious disease control and public health threat assessment. Technologies such as flow cytometry can provide clinicians with the early information, they need for appropriate antibiotic treatment. At the same time, cytometry platforms facilitate the measurement of antibiotic-resistant bacteria in environments impacted by human activities, enabling assessment of their impact on watersheds and soils. This review focuses on the latest applications of flow cytometry for the detection of pathogens and antibiotic-resistant bacteria in both clinical and environmental samples. Novel antimicrobial susceptibility testing frameworks embedding flow cytometry assays can contribute to the implementation of global antimicrobial resistance surveillance systems that are needed for science-based decisions and actions.

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“…A potential solution to this problem is the use of single-cell specific ASTs, which address the heterogeneity problem while also offering higher throughput by evaluating the effect of antimicrobials directly on cells, rather than relying on secondary markers, such as growth of an entire culture. Multiple such candidate ASTs have been proposed, enabled by platforms such as flow cytometry 10 , Raman spectroscopy 11,12 , fluorescent probes in droplet microfluidic devices 13 , impedence cytometry 14 and others. Further opportunity in single-cell ASTs comes from their integration with widefield microscopy [15][16][17][18] , which further increases throughput by enabling real-time simultaneous monitoring of large numbers of individual cells.…”

Section: Introductionmentioning

confidence: 99%

Deep Learning and Single Cell Phenotyping for Rapid Antimicrobial Susceptibility Testing

Zagajewski

Turner

Feehily

et al. 2022

Preprint

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The rise of antimicrobial resistance (AMR) is one of the greatest public health challenges, already causing up to 1.2 million deaths annually and rising. Current gold-standard antimicrobial susceptibility tests (ASTs) are low-throughput and can take up to 48 hours, with implications for patient care. We present advances towards a novel, rapid AST, based on the deep-learning of single-cell specific phenotypes directly associated with antimicrobial susceptibility in Escherichia coli. Our models can reliably (80% single-cell accuracy) classify untreated and treated susceptible cells, across a range of antibiotics and phenotypes - including phenotypes not visually distinct to a trained, human observer. Applying models trained on lab-reference susceptible strains to clinical isolates of E. coli treated with ciprofloxacin, we demonstrate our models reveal significant (p<0.001) differences between resistant and susceptible populations, around a fixed treatment level. Conversely, deploying on cells treated with a range of ciprofloxacin concentrations, we show single-cell phenotyping has the potential to provide equivalent information to a 24-hour growth AST assay, but in as little as 30 minutes.

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Same-day confirmation of infection and antimicrobial susceptibility profiling using flow cytometry

Cited by 9 publications

References 23 publications

Bridging Flocculation of a Sterically Stabilized Cationic Latex as a Biosensor for the Detection of Microbial DNA after Amplification via PCR

Bridging Flocculation of a Sterically Stabilized Cationic Latex as a Biosensor for the Detection of Microbial DNA after Amplification via PCR

Current and Future Flow Cytometry Applications Contributing to Antimicrobial Resistance Control

Deep Learning and Single Cell Phenotyping for Rapid Antimicrobial Susceptibility Testing

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