Testing Antibiotic Resistance with a Fast, Inexpensive and Easy Method

Researchers at EPFL and Vrije Universiteit Brussel have developed a novel and highly efficient method for rapid antibiotic susceptibility testing using optical microscopy. The technique, called Optical Nanomotion Detection, is extremely rapid, single-cell sensitive, label-free, and requires only a basic traditional optical microscope, equipped with a camera or a mobile phone.

“We have developed a technique in our laboratories that allows us to obtain an antibiogram within 2-4 hours – instead of the current 24 hours for the most common germs and one month for tuberculosis,” says Dr Sandor Kasas at EPFL. Professor Ronnie Willaert at Vrije Universiteit Brussel adds: “Our technique is not only faster but also simpler and much cheaper than all those existing now.”

Antibiotic resistance happens when bacteria develop the ability to defeat the drugs designed to kill them. It has now grown into a global public health issue. It was responsible for at least 1.27 million deaths worldwide in 2019 while being involved in nearly five million deaths. Every year, the US sees almost three million antimicrobial-resistant infections, with the cost of treating the six most common ones at over $4.6 billion. The EU sees almost 700,000 cases each year, which cost it an estimated €1.5 billion.

Antibiotic sensitivity testing (AST) uses culture methods that expose bacteria to antibiotics, or genetic methods to determine if bacteria possesses genes that confer resistance. Typical ASTs last up to 24 hours or even longer for slow-growing bacteria – a timeframe that can mean life or death in a clinical setting. There have been some faster ASTs developed in recent years, but they tend to be complex, needing sophisticated and expensive equipment.

Now, researchers led by Kasas and Willaert have developed a fast, cheap, and widely accessible method based on optical microscopy that can perform an AST with single-cell sensitivity and without needing to attach or label bacteria. The technique uses a basic, conventional optical microscope, a camera or mobile phone, and dedicated software. The joint research project is published in PNAS.

The new technique is called optical nanomotion detection (ONMD), and involves the monitoring of nanoscale vibrations of single bacterial before and while being exposed to antibiotics. The monitoring is performed with a basic optical microscope, a video camera or a mobile phone.

The ONMD technique monitors the microscopic oscillations of bacterial cells (nanomotion) that characterize living organism and can be considered as a “signature of life”. Indeed, nanomotion lasts as long the organism is alive but stops immediately when it is dead. In the ONMD technique, bacterial nanomotion is recorded in a movie in which all individual cell displacements are monitored with sub-pixel resolution.

The researchers used ONMD to successfully detect the sensitivity of numerous bacteria to antibiotics. Escherichia coli, Staphylococcus aureus, Lactobacillus rhamnosus, and Mycobacterium smegmatis (a non-pathogenic bacterial model for tuberculosis) sensitivities to the antibiotics ampicillin, streptomycin, doxycycline, and vancomycin was determined in less than two hours.

The ONMD not only monitors the bacteria life-death transitions upon exposure to different antibiotics but also highlights changes in the bacteria’s metabolism caused by the availability of nutrients. The tests showed that ONMD can assess the sensitivity or resistance of bacterial cells to antibiotics in a simple and rapid way by monitoring cellular oscillations.

The authors state: “The simplicity and efficiency of the method make it a game-changer in the field of AST” as it can be applied to a wide range of bacteria, which has significant implications for clinical and research applications.

Other contributors

University of Lausanne (UNIL)

Funding

Swiss National Science Foundation (SNSF)

Research Foundation – Flanders (FWO)

Belgian Federal Science Policy Office (Belspo)

European Space Agency (ESA)

Reference

Maria I. Villalba, Eugenia Rossetti, Allan Bonvallat, Charlotte Yvanoff, Vjera Radonicic, Ronnie G. Willaert, Sandor Kasas. Simple optical nanomotion method for single-bacterium viability and antibiotic response testing. PNAS 28 April 2023. DOI: 10.1073/pnas.2221284120 

Source: École Spéciale de Lausanne

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Researchers Develop Rapid Test for Salmonella Food Contamination

McMaster University researchers have developed a rapid and inexpensive test for Salmonella contamination in chicken and other food — one that’s easier to use than a home COVID test.

The test, described in a new paper in the journal Angewandte Chemie, could improve food safety, reduce the cost of processing fresh poultry and other foods, and help to limit broad recalls to batches that have specifically been identified as contaminated.

The researchers have shown that the test provides accurate results in an hour or less without the need for accessories or a power source, compared to today’s monitoring through lab cultures, which require at least a full day to produce results.

Once scaled up and made available commercially, the new test could be a significant boon to poultry processors, for whom salmonella is among the most significant contamination risks.

The test would also be beneficial for ensuring the safe processing of other foods that are particularly vulnerable to salmonella, such as eggs, dairy products and ground beef.

A single major poultry processor performs tens of thousands of salmonella lab tests each year. Reducing or even eliminating the need for overnight lab cultures would represent significant savings and make it easier to identify contamination earlier in the process.

“Anyone can use it right in the setting where food is being prepared, processed or sold,” says study co-author Yingfu Li, a professor of Biochemistry and Chemical Biology who leads McMaster’s Functional Nucleic Acids Research Group.

Protecting the public from salmonella is a high priority for food producers, retailers and regulators alike, since it is one of the most common and serious forms of food-borne infection, causing 155,000 deaths globally every year.

What makes the test work is a new synthetic nucleic acid molecule, developed at McMaster. For the test, the molecule is sandwiched between microscopic particles such as gold.

The test platform lines the inside of the tip of a pipette and begins to work when a liquefied sample of the food being tested is drawn inside the tube.

If salmonella bacteria are present, they cut through the particles, allowing the molecule to escape.

When the solution is dropped onto a paper test strip, the presence of salmonella shows as a visible shade of red, thanks to a new form of biosensor, also created by the McMaster team. The greater the concentration of salmonella, the brighter the colour appears.

The new technology has been developed with support from the non-profit research organization Mitacs, and Toyota Tsusho Canada Inc., an indirect subsidiary of Toyota Tsusho Corporation in Japan, which plans to develop the innovation for commercial use.

The research is part of an ongoing, broader effort to establish McMaster and its Global Nexus for Pandemics and Biological Threats as a centre for the development of real-time sensors, pathogen-repellent materials and other products that improve food safety.

“This is very important to us in the development of our food-testing program,” says co-author Tohid Didar, an associate professor of Mechanical Engineering and Canada Research Chair in Nano-biomaterials.

Li, Didar and Filipe authored the paper with postdoctoral research fellow Jiuxing Li, PhD student and Vanier Scholar Shadman Khan, and research associate Jimmy Gu.

Reducing illness and food waste aligns with Toyota Tsusho Canada’s values, explained Toyota Tsusho Canada Inc. President Grant Town.

“Our goal is to help bring proven research from the lab to the marketplace, where it can benefit society,” Town says. “Reducing the risk of illness while also cutting food waste will benefit everyone, and Toyota Tsusho Canada sees this as a great opportunity.”

Reference

A Simple Colorimetric Au-on-Au Tip Sensor with a New Functional Nucleic Acid Probe for Food-borne Pathogen Salmonella typhimurium. Li, J., Khan, S., Gu, J., Filipe, C. D. M., Didar, T. F., Li, Y., Angew. Chem. Int. Ed. 2023, e202300828; Angew. Chem. 2023. https://doi.org/10.1002/anie.202300828. 

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Microbiology Poems, Courtesy of ChatGPT

ChatGPT is a natural language processing tool driven by AI technology that allows you to have human-like conversations and much more with the chatbot. The language model can answer questions and assist you with tasks like composing emails, essays, and code.

I was curious as to the power of using ChatGPT to write an essay about microbiology. But to have fun, I asked the chatbot to write poems about microbiology and more specifically, about rapid microbiological methods. Within 5 seconds, surprisingly accurate (and humorous) poems were written, which I have reproduced below. My disclaimer: ChatGPT must obtain its information from somewhere in public domain (e.g., Wikipedia, books, news articles, scientific journals), and as such, the poems presented below may have been generated from prior published work. 

A Poem About Rapid Microbiological Methods (my favorite)

In labs of science, with cutting-edge tech,

Rapid microbiological methods, a breakthrough, a check,

A faster way, a modern stride,

To detect and identify, with precision, worldwide.

Gone are the days of lengthy waits,

For microbial results, sealing fates,

Rapid methods, swift and true,

Reducing time, a remarkable coup.

PCR, MALDI-TOF, and more,

Using automation, they soar,

High-throughput screening, in a flash,

Rapid methods, a time-saving splash.

From food safety to clinical care,

Rapid methods, a boon, so rare,

Detecting pathogens, with speed,

Saving lives, meeting the need.

In pharmaceuticals, in water, in air,

Rapid methods, with accuracy, they dare,

To identify contaminants, in a blink,

Empowering industries, with a new link.

Quality control, process monitoring,

Rapid methods, an efficient adorning,

Ensuring safety, reducing risk,

Microbial analysis, at a brisk.

But validation, a crucial part,

Ensuring reliability, from the start,

Robustness, accuracy, and precision,

Validating rapid methods, a mission.

So, here's to rapid microbiological ways,

Advancing science, in modern days,

Bringing speed, efficiency, and control,

Rapid methods, a boon to behold.

A Poem About Microbiology (#1)

Microbes unseen, a world so small,

Invisible to the naked eye, yet rule them all.

They dwell in the air, in water, in soil,

Microbes in every corner, a hidden coil.

Bacteria, fungi, viruses, and more,

Microbiology's wonders to explore.

They're the masters of decomposition,

Breaking down the organic with precision.

In labs, scientists study their ways,

Peering through microscopes, in a curious gaze.

Culturing, identifying, studying their traits,

Unraveling mysteries, their intricate debates.

From penicillin to vaccines and beyond,

Microbes offer solutions, a treasure to be found.

They can cure, they can harm,

Infectious diseases can cause alarm.

But microbiology is a field of great might,

Harnessing nature's power, shining bright.

Understanding the tiny world of life,

Bringing knowledge, reducing strife.

In food, in health, in the environment,

Microbes play a role, so significant.

From fermentation to biotechnology,

Microbiology's impact, a boundless sea.

So let us marvel at the unseen,

The microbial world, a wonder so keen.

A realm of diversity, complexity,

Microbiology's beauty, a true delicacy.

A Poem About Microbiology (#2)

In a microscopic world unseen,

Where wonders dwell, both small and keen,

Microbes thrive, in vast array,

A realm of life, in a tiny display.

Bacteria, tiny cells, so diverse,

In shapes and sizes, they immerse,

Some are round, some are rod-like,

In a microscopic world, they strike.

Viruses, smaller still, but mighty,

Intracellular hijackers, acting slyly,

Invading host cells, causing disease,

Microbiology's puzzle, a complex tease.

Fungi, with their branching threads,

Mycelium network, like nature's threads,

Mushrooms, molds, and yeasts, they grow,

A world of fungi, both friend and foe.

From gut to skin, from air to soil,

Microbes thrive, in their daily toil,

They decompose, they ferment,

In symbiosis, they are inherent.

They aid digestion, break down waste,

In bioremediation, they show their taste,

In brewing, baking, and making cheese,

Microbes play a role, with utmost ease.

In labs, scientists study their ways,

Unraveling secrets, through endless days,

Identifying species, understanding their traits,

Microbiology's mysteries, with endless rates.

They create antibiotics, vaccines, and more,

Innovations galore, a microbial store,

Solving problems, finding cures,

Microbes, a blessing that endures.

So let's appreciate the microscopic view,

The world of microbiology, a wonder so true,

A realm of life, both small and grand,

Microbes, a marvel, forever to stand.

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Label-free Droplet-based Methods Improve Rapid Screening and Sorting of Bacteria

Effective, accurate and quick ways to screen and sort microbes are in short supply. Most methods available now rely on additional labeling steps to sort bacteria, which are typically time-consuming and cannot work well for industrial-scale breeding. 

The need for accuracy and speed are met using a label-free droplet-based integrated microfluidic platform that screens bacterial growth based on phenotype, or observable characteristics. Better and quicker screening of bacterial growth can have considerable effects on medical, pharmaceutical and agricultural industries. 

Screening of bacteria by their observable characteristics may not seem possible, but the researchers from the Qingdao Institute of Bioenergy and Bioprocess Technology (QIBEBT) of the Chinese Academy of Sciences (CAS) determined how to best do this using a microfluidic system and the autofluorescent properties of bacteria.

The results were published in Sensors and Actuators B: Chemical on March 22.

The main functions of this microfluidic platform are organized in a box, which houses the "detection area" the droplets will go through, along with photomultiplier tubes (PMT) and an optical fiber inserted through the chip. A light signal is transmitted to the PMT, and is sorted based on the intensity. There are parameters that can be set both high and low to sort out the target droplets easier.

"A flexible, label-free droplet-based detector allowing bacterial growth phenotype screening may help to expand the scope of rapid bacteria screening in various applications, especially in high-quality industrial breeding," said GE Anle, researcher and first author of the study. 

The researchers determined by using PMT that high densities of bacteria such as E. coli were able to be divided into a sorting channel when exposed to an 800V wave pulse for 5 to 20 milliseconds. Any droplets that did not make it to sorting thresholds were left to fall into the waste channel, and these were typically empty, or negative, droplets. Positive droplets, or droplets with bacteria in them, got deflected into a sorting channel since they were within the given threshold. 

Results from the study indicated sorting efficiency of 95.3% when it comes to cell-containing (positive) droplets, and 91.7% of empty (negative) droplets successfully made it to the waste channel. 

"We anticipate that the mini integrated microfluidic system will serve as a useful platform for further subsequent analyses, including antibiotic resistance, metabolic analysis and industrial strains growth phenotype screening," said DIAO Zhidian, researcher and co-author of the study. 

The microfluidic platform has many advantages compared to the way bacterial growth phenotype screening is done currently. Using an optical fiber to transmit light eliminates the need for a complex lighting system, and most of the core functions are fully integrated into the box structure. 

"The use of determining phenotype using light properties but not relying on fluorescent labels is important, especially because many different cell types may not be compatible with sorting through the use of fluorescent labeling," said corresponding author Prof. MA Bo, from Single-Cell Center of QIBEBT. "The last big advantage is the droplet method: isolating individual bacteria in a droplet can allow for better chances of proliferation, which is of utmost importance in fields involving large-scale industrial breeding of bacteria." 

"We have developed Raman-based flow cytometry tools, such as FlowRACS which improves accuracy, throughput, and stability in profiling dynamic metabolic features of cells, to screen 'high-yield' strains rapidly without the need to label the cell with fluorescence probes. This newly developed microfluidic platform can further screen the 'fast-growing' strains rapidly, so as to achieve the goal of industrial microbial breeding," said Prof. XU Jian, the head of Single-Cell Center of QIBEBT. "Next step, we will further develop the key technologies and equipment platform for single-cell breeding to support the development of industrial biotechnology and synthetic biology."

Source: Chinese Academy of Sciences

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Simplified Screening of Individual Bacterial Species in Biosamples

In medical research and diagnostics, the microbiome, i.e. the microbial colonisation of the intestine, is increasingly gaining attention. A stool sample can be used to precisely analyse the complex microbial ecosystem of the gut. There are basically two methods for this: traditional cultivation on plates with specific culture media or the quite expensive DNA analysis of the stool sample. Both methods on their own are unsatisfactory when it comes to quickly detecting a certain bacterial species in the sample. Researchers from the research training group (RTG) Translational Evolutionary Research (TransEvo) at Kiel University (CAU) have developed a rapid and robust screening method in order to specifically detect and cultivate lactobacilli, bifidobacteria and Bacteroides in stool samples. They published the results in the journal Current Microbiology. "Our method is less expensive and faster than alternative methods usually used to isolate and identify bacteria in complex microbiological samples," explained first author Sofia Borges, a PhD student at the Department of Microbiology and Biotechnology at the Max Rubner-Institut in Kiel. "The method is particularly suitable for screening for certain bacterial species for which there is no exclusive culture medium," added Professor Charles Franz, head of the institute and Associate Professor at the Faculty of Agricultural and Nutritional Sciences at Kiel University. "With this method, we are saving ourselves lengthy procedures for singling out pure bacterial species from potentially non-pure colonies and identifying them."

Bacterial culture is often not selective

Bacterial culturing is the cultivation of microorganisms on a culture medium under controlled conditions, such as temperature. The culture medium that is selected depends on which bacterial species you are looking for in the sample. Selective growth media promote the growth of specific species, while the growth of other species contained in the sample is inhibited. If the species you are looking for is in the sample, it will grow into a colony. That is the ideal scenario. However, the culture media are often not exclusively selective for only one species, but also allow a few other bacterial species to grow.

"In this study, we focused on bifidobacteria, lactobacilli and Bacteroides," said Borges. Bifidobacteria and lactobacilli were chosen because they are relevant to gut health, but are usually not found in large numbers in the intestine. Therefore, even with partially selective media, it is sometimes not possible to cultivate these bacteria. Bacteroides were included in the study as an example of a gram-negative species that is also relevant for gut health and is being further studied by the co-authors of the study.

Detection of pure colonies despite lack of purification

The principle of the method is based on cultivation using three different selective media, DNA extraction, PCR analysis of a specific gene and sequencing. Six stool samples from healthy individuals were used to test this method. Cultivation took place in an anaerobic chamber (without oxygen) for 48 hours at 37 degrees Celsius. Afterwards, well-separated individual colonies were selected for the isolation of the bacteria. The individual colonies were examined according to the study protocol. It was possible to identify the bacterial species contained in all 180 colonies. Most colonies could be assigned to a single species, even though the selective media used not only favoured the growth of the target bacteria but also allowed a few other species to grow. "Some of our colonies contained up to three different species of bacteria. However, we were pleasantly surprised to find that most colonies were pure, despite minimal cultivation and no purification by repeatedly streaking out colonies," explained Borges.

Her working group leader Charles Franz summed up as follows: "Our new method makes it possible to gain insight into the purity of the colonies present on agar plates and to accurately identify the bacteria they contain. It can therefore be useful in providing a speedy, cost-effective and robust overview of bacteria recovered from complex microbiological samples before selecting them for further study."

Reference:

Borges, A.S.G., Basu, M., Brinks, E. et al. Fast Identification Method for Screening Bacteria from Faecal Samples Using Oxford Nanopore Technologies MinION Sequencing. Curr Microbiol 80, 101 (2023). https://doi.org/10.1007/s00284-023-03201-7

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Scientists Apply Raman Quantitative 3D Imaging to Microbial Monitoring

Microorganisms are important contributors to the deep-sea sulfur cycle. However, in-situ detection of deep-sea microorganisms faces great challenges due to the extreme complexity of the deep-sea environment, the difficulty of sampling, isolation and cultivation of microorganisms, and the lack of near real-time nondestructive monitoring methods for microbial sulfur metabolism.

To help with this challenge, we can turn to Raman (spectroscopy, not the noodle). 😊

Recently, a research team led by Prof. Zhang Xin and Prof. Sun Chaomin from the Institute of Oceanology of the Chinese Academy of Sciences (IOCAS) achieved long-term, near real-time, non-destructive microbial monitoring through 3D quantitative imaging based on the confocal Raman technology.

The study was published in Microbiology Spectrum. 

Currently, the process of elemental sulfur production is mainly studied by classical biological and chemical methods, such as X-ray near-edge absorption spectroscopy, high-performance liquid chromatography, transmission electron microscopy, ion chromatography and chemometrics. However, these methods are mainly used to investigate the metabolism of microorganisms at specific time points through sampling, and cannot continuously monitor their metabolic processes on time scales without destroying the cells.

Moreover, some of these methods have complicated sample preparation, which will destroy the in-situ conditions of cells. They may also result in uneven sampling and contamination, making it difficult to achieve continuous in-situ observation.

The confocal Raman 3D imaging is low-cost, rapid, label-free and non-destructive, and has the potential to perfectly combine qualitative, quantitative and visualization.

To demonstrate the potential of this technique, Prof. Zhang's team constructed Raman 3D quantitative in-situ analysis method for microbial communities on solid substrates. It combined optical visualization with Raman quantitative analysis, and could quantitatively characterize the microbial metabolic processes in both temporal and spatial dimensions non-destructively.

The technique has been applied to the in-situ monitoring of the sulfur metabolic processes of the deep-sea cold-seep bacterium E. flavus 21-3. Volume calculations and ratio analysis based on Raman 3D imaging have quantified the growth and metabolism of the microbial colony in different environments. It uncovered the unknown details of microbial growth and metabolism, and provided an important technical support for clarifying the causes of the widely distributed elemental sulfur in deep-sea cold seep.

"To our knowledge, this is the first in-situ nondestructive technique to attempt long-term monitoring of microbial growth and metabolism in solid medium. It supports to rapidly identify metabolites, infer biological pathways, screen the optimal metabolic conditions of microorganisms, and compare the elemental sulfur production rate of different strains," said Prof. Zhang.

"The successful application of this technology not only demonstrates the potential of the method for future visualization and quantitative analysis of microbial processes in situ, but also provides new ideas for studying microorganisms attached to solid surfaces in natural environment," said Prof. Sun.

Reference:

Wanying He et al, Study of Microbial Sulfur Metabolism in a Near Real-Time Pathway through Confocal Raman Quantitative 3D Imaging, Microbiology Spectrum (2023). DOI: 10.1128/spectrum.03678-22

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