Label-free SERS Technology Realizes Rapid Identification of Beer Spoilage Bacteria

In a study published in Analytical Methods, a research group led by LI Bei from the Changchun Institute of Optics, Fine Mechanics and Physics (CIOMP) of the Chinese Academy of Sciences (CAS) proposed rapid detection of beer spoilage bacteria based on label-free surface-enhanced Raman spectroscopy (SERS) technology.

Lactic acid bacteria are common spoilage bacteria in beer and need to be monitored and controlled at all stages of beer production. Traditional spoilage bacteria detection methods are time-consuming and cannot meet the demand for real-time, in-situ, rapid detection during the production process.

Raman spectroscopy has been widely used for microbial detection due to its fast, non-destructive and accurate properties, but conventional Raman spectroscopy has the disadvantage of poor signal-to-noise ratio, which affects the accuracy of microbial identification.

Compared with conventional Raman spectroscopy, the SERS technique has a stronger and more sensitive signal and is well suited to the detection of beer spoilage bacteria. Furthermore, the label-free SERS technique is ideal for commercialization due to its low cost and good results.

In this study, the researchers improved the existing process for the preparation of label-free SERS silver nanoparticles (AgNPs). The effect of the AgNPs@KCl agglomeration effect on SERS enhancement was investigated. Eight species of beer spoilage bacteria produced during the beer brewing process were identified by SERS.

The researchers further investigated the effect of the method on the final identification rate by combining the t-distributed stochastic neighbor embedding (t-SNE) dimensionality reduction analysis algorithm, Support Vector Machine (SVM), k-NearestNeighbor (KNN) and Linear Discriminant Analysis (LDA) machine learning algorithms. All three machine learning algorithms achieved an accuracy of around 90% and performed well in identifying beer spoilage bacteria.

In the stability analysis and mixing tests, two known spoilage bacteria were mixed with pure beer and incubated at constant temperature for a period of time to identify the bacteria in the beer. The two spoilage bacteria were successfully detected in the samples and had good spectral stability.

According to the final validation study, the technique can indeed identify the target spoilage bacteria from the simulated samples, which is of great significance to the rapid identification of spoilage bacteria in beer brewing process.

Reference

Lindong Shang et al, Rapid detection of beer spoilage bacteria based on label-free SERS technology, Analytical Methods (2022). DOI: 10.1039/D2AY01221A 

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RT-PCR Could be a More Rapid Test for C. Difficile on Environmental Surfaces

There might be a new way to detect Clostridiodes difficile on surfaces rapidly, which could be especially crucial in hospital settings.

A team, led by Rachel J. Grainger, Department of Clinical Microbiology, RCSI Education and Research Centre, Royal College of Surgeons in Ireland, Beaumont Hospital, compared the detection of C. difficile by RY-PCR to culture-based strategies and determined which is more sensitive and specific in a clinical environment.

Detecting C. difficile

Detection of C. difficile from the environment is generally done using bacterial culture, which can take several days to receive results. In addition, environmental surveillance is not usually done routinely and only conducted during outbreak investigations or as part of research.

“Environmental surveillance for Clostridioides difficile is challenging,” the authors wrote. “There are no internationally agreed recommendations on which method should be used when environmental surveillance is undertaken.”

Currently, culture-based tests are the most commonly used testing methods but can take 48 hours for results. Molecular methods also offer lower TATs, but these methods are not yet optimized.

There is a need for more rapid methods to detect C. difficile, which could aid in preventing infections.

“Unlike culture for CD, which takes too long, molecular methods such as PCR have potential in monitoring the patient environment in hospitals for CD and might be used to provide reassurance to patients and hospitals before patients are admitted to a hospital bed,” the authors wrote.

The study included 44 near patient areas of patients who are C. difficile positive, each 1 sampled using contact plates and moistened flocked swabs. The investigators took environmental samples over a 6-month period in an adult 820-bed tertiary referral hospital from the floor of the room, the bed rail, the tray table, the call bell, the mattress, the toilet floor, the toilet flush handle, and the internal bathroom door handle.

Finding Results From Samples

There were 352 samples taken using flocked swabs, resulting in 59 positive samples. This was more than the 35 positive samples found with alcohol treated and sub-cultured onto C. difficile selective agar (P = 0.01).

Moreover, there were 23 samples positive using both culture methods, 36 samples positive using the CHROMID agar only, and 12 samples positive using the alcohol treatment and culture method only.

There were 71 samples culture-positive for C. difficile using the flocked swab method compared to 29 samples using the contact plates.

The results also show 5.43% (n = 19) of samples were positive using both methods, 14.86% (n = 52) were positive only by the flocked swab and culture-method, and 2.86% (n = 10) were positive only by contact plates.

Finally, 15.14% (n = 53) of samples were positive using both the tcdB RT-PCR assay and the culture-based method, while 4.86% (n = 17) were positive using the RT-PCR negative for tcdB and 7.43% (n = 26) were RT-PCR positive for tcdB but culture-negative.

The investigators also analyzed positive samples taken from specific area of the infected patient areas.

The surface with C. difficile most frequently found on it was mattresses (n = 15; 36 %), followed by room floors (n = 14; 29 %) and toilet floors (n = 9; 31 %) using a flocked swab followed by RT-PCR for tcdB.

However, after using the flocked swab sampling technique and culture, the investigators detected C. difficile from mattresses (n = 11; 26 %), toilet floors (n = 8; 28 %), and room floors (n = 13; 27 %). When they used contact plates, they detectedC. difficile most frequently from the room floor (n = 5; 10 %), toilet floor (n = 7; 24 %), and the arm of armchair (n = 4; 27 %).

The results show detection using moistened flocked swabs followed by RT-PCR or culture resulted in more C. difficile detected compared to using the contact pates. The sensitivity of a RT-PCR assay for tcdB was 76% compared to the culture methods, while the specificity was 91%.

“Despite the lower sensitivity and specificity, RT-PCR could potentially offer a more rapid and practical alternative,” the authors wrote. “While culture picked up more positive samples for CD, PCR detected about three-quarters of the positive samples but the results from PCR were available within hours and not days.”

The investigators suggested future research should focus on confirming the role of PCR in the prevention and control of C. difficile in hospitals.

The study, “A comparison of culture methods and polymerase chain reaction in detecting Clostridioides difficile from hospital surfaces,” was published online in the Journal of Medical Microbiology.

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Diagnostic Aid Rapidly Identifies Respiratory Pathogens in Critically ill Children

A molecular diagnostic aid provides reliable and fast respiratory pathogen identification in mechanically ventilated children with pneumonia.

A diagnostic aid based on polymerase chain reactions (PCR), that uses a 52-pathogen custom array card, has been found to provide both rapid (compared to blood culture) and reliable information on respiratory infections in critically ill, mechanically ventilated children, according to a study by UK researchers.

Respiratory tract infections are responsible for a large number of admissions to paediatric intensive care units. Moreover, an intensive care unit is unique environment and for which clinicians often make decisions to use antibiotics with some degree of diagnostic uncertainty. This was clearly illustrated in one study of paediatric intensive care unit children, where despite most critically children receiving antimicrobial therapy, infection was often not microbiologically confirmed. While in many cases, respiratory infections are viral in nature, it is necessary to utilise methods such as quantitative PCR, as a diagnostic aid to identify the presenting pathogens. In fact, a recent study in adults found that multiplex bacterial PCR examination of bronchoalveolar lavage, reduced the duration of inappropriate antibiotic therapy of patients admitted to hospital with pneumonia and who were at risk of Gram-negative infection. 

In the current study, researchers made use of the TaqMan Array Card (TAC) as a diagnostic aid which is a microfluidic quantitative PCR system comprising of 384 wells containing pre-aliquoted customised primer and probe combinations. The aid has been previously shown to be of value in supporting ventilator-associated pneumonia (VAP) diagnosis in adults. Nevertheless, it has not been examined in critically ill children and therefore, the aim of the present study was to assess the utility of TAC to identify bacterial and fungal respiratory pathogens in critically ill children with suspected community acquired pneumonia or VAP. The study recruited children ≤ 18 years of age and if they were mechanically ventilated and had commenced or were commencing antimicrobial therapy for a lower respiratory tract infection. The researchers determined the sensitivity and specificity of TAC to detect bacterial and fungal pathogens causing lower respiratory tract infections and the time to a result provided by TAC compared to standard microbiology cultures. Secondary objectives included a description of the micro-organisms detected by TAC but not by microbiology culture as well as the impact of TAC on antimicrobial decision-making.

Diagnostic aid and outcomes

A total of 100 children with a median age of 1.2 years (58% male) were included in the study and of whom, 80 had suspected community acquired pneumonia and the remainder, hospital acquired pneumonia.

Bacteria were detected more frequently on TAC compared to microbiology cultures (57% vs 18%, p < 0.001)) and In addition, TAC also identified more fungi (17% vs 2%, p < 0.001).

For the detection of bacterial and fungal species, TAC had a sensitivity of 89.5% (95% CI 66.9 – 98.7) and a specificity of 97.9% (95% CI 97.2 – 98.5). The median time to obtain a result for the diagnostic aid was 25.8 hours compared to 110.4 hours for microbiological cultures and overall, TAC was significantly quicker for both positive and negative results (p < 0.001).

Finally, consultants reported a change of prescription in 47% of cases based upon TAC results. Antimicrobial therapy duration was reduced or stopped in 26% of children, extended in16% and the spectrum of treatment was broadened in 17% of cases and reduced in 17%.

The authors concluded that as a diagnostic aid, TAC can be used to reliably detect pathogens quicker than routine culture in critically ill children with suspected lower respiratory tract infections and called for future studies to incorporate antimicrobial decision support and economic analysis.

Citation

Clark JA et al. The rapid detection of respiratory pathogens in critically ill children. Crit Care, 2023

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Highly Accurate Test for Common Respiratory Viruses Uses DNA as ‘Bait’

A new test that ‘fishes’ for multiple respiratory viruses at once using single strands of DNA as ‘bait’, and gives highly accurate results in under an hour, has been developed by Cambridge researchers.

The test uses DNA ‘nanobait’ to detect the most common respiratory viruses – including influenza, rhinovirus, RSV and COVID-19 – at the same time. In comparison, PCR (polymerase chain reaction) tests, while highly specific and highly accurate, can only test for a single virus at a time and take several hours to return a result.

While many common respiratory viruses have similar symptoms, they require different treatments. By testing for multiple viruses at once, the researchers say their test will ensure patients get the right treatment quickly and could also reduce the unwarranted use of antibiotics.

In addition, the tests can be used in any setting, and can be easily modified to detect different bacteria and viruses, including potential new variants of SARS-CoV-2, the virus which causes COVID-19. The results are reported in the journal Nature Nanotechnology.

The winter cold, flu and RSV season has arrived in the northern hemisphere, and healthcare workers must make quick decisions about treatment when patients show up in their hospital or clinic.

“Many respiratory viruses have similar symptoms but require different treatments: we wanted to see if we could search for multiple viruses in parallel,” said Filip Bošković from Cambridge’s Cavendish Laboratory, the paper’s first author. “According to the World Health Organization, respiratory viruses are the cause of death for 20% of children who die under the age of five. If you could come up with a test that could detect multiple viruses quickly and accurately, it could make a huge difference.”

For Bošković, the research is also personal: as a young child, he was in hospital for almost a month with a high fever. Doctors could not figure out the cause of his illness until a PCR machine became available.

“Good diagnostics are the key to good treatments,” said Bošković, who is a PhD student at St John’s College, Cambridge. “People show up at hospital in need of treatment and they might be carrying multiple different viruses, but unless you can discriminate between different viruses, there is a risk patients could receive incorrect treatment.”

PCR tests are powerful, sensitive and accurate, but they require a piece of genome to be copied millions of times, which takes several hours.

The Cambridge researchers wanted to develop a test that uses RNA to detect viruses directly, without the need to copy the genome, but with high enough sensitivity to be useful in a healthcare setting.

“For patients, we know that rapid diagnosis improves their outcome, so being able to detect the infectious agent quickly could save their life,” said co-author Professor Stephen Baker, from the Cambridge Institute of Therapeutic Immunology and Infectious Disease. “For healthcare workers, such a test could be used anywhere, in the UK or in any low- or middle-income setting, which helps ensure patients get the correct treatment quickly and reduce the use of unwarranted antibiotics.”

The researchers based their test on structures built from double strands of DNA with overhanging single strands. These single strands are the ‘bait’: they are programmed to ‘fish’ for specific regions in the RNA of target viruses. The nanobaits are then passed through very tiny holes called nanopores. Nanopore sensing is like a ticker tape reader that transforms molecular structures into digital information in milliseconds. The structure of each nanobait reveals the target virus or its variant.

The researchers showed that the test can easily be reprogrammed to discriminate between viral variants, including variants of the virus that causes COVID-19. The approach enables near 100% specificity due to the precision of the programmable nanobait structures.

“This work elegantly uses new technology to solve multiple current limitations in one go,” said Baker. “One of the things we struggle with most is the rapid and accurate identification of the organisms causing the infection. This technology is a potential game-changer; a rapid, low-cost diagnostic platform that is simple and can be used anywhere on any sample.”

A patent on the technology has been filed by Cambridge Enterprise, the University’s commercialisation arm, and co-author Professor Ulrich Keyser has co-founded a company, Cambridge Nucleomics, focused on RNA detection with single-molecule precision.

“Nanobait is based on DNA nanotechnology and will allow for many more exciting applications in the future,” said Keyser, who is based at the Cavendish Laboratory. “For commercial applications and roll-out to the public we will have to convert our nanopore platform into a hand-held device.”

“Bringing together researchers from medicine, physics, engineering and chemistry helped us come up with a truly meaningful solution to a difficult problem,” said Bošković, who received a 2022 PhD award from Cambridge Society for Applied Research for this work.

The research was supported in part by the European Research Council, the Winton Programme for the Physics of Sustainability, St John’s College, UK Research and Innovation (UKRI), Wellcome, and the National Institute for Health and Care Research (NIHR) Cambridge Biomedical Research Centre.

Reference:

Filip Bošković et al. ‘Simultaneous identification of viruses and viral variants with programmable DNA nanobait.’ Nature Nanotechnology (2022). DOI: 10.1038/s41565-022-01287-x

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