Hoping to help patients and
prevent the spread of drug-resistant bacteria
by identifying the causative microbes of bloodstream infections

Research companions with broad research horizons and a diverse background are an attraction of Hitachi

Yanagawa：In college, I majored in electronic engineering and was studying electronic circuitry. I conducted research on highly reliable circuits for satellites in the Japan Aerospace Exploration Agency (JAXA) for 5 years as my master’s and doctor’s program. I was also involved in the launching of satellites. These satellites would continue flying for several years even after my graduation. I had the hope of manufacturing something tangible that would last over a long period using pioneering technology. I joined Hitachi because I heard from a senior classmate that it had one of the largest research laboratories among private companies.

After I joined Hitachi, I was assigned to the development of DRAM (Dynamic Random Access Memory), which is a semiconductor memory device. About 7 years ago, I was transferred to the Biosystems Research Department, and now I am developing genetic testing technology. The business environment changes with time, but I consider the diverseness of research fields from semiconductors to biosystems, which permits me to make use of my skills even if I am transferred to other fields, a characteristic of Hitachi.

Shimizu：In my undergraduate and graduate schools of chemistry, I majored in biochemistry. I studied genetic engineering to enhance the function of genes that leads to mRNA vaccines against like COVID-19 and nucleic acid medicines. I came to know that Hitachi is also engaged in the biological field as I saw Hitachi’s advertisement for intern recruitment proposing monitoring of biological drugs as a research theme. After a 3-week internship, I renewed my aspiration for research in the field of biology and joined Hitachi. This is my fourth year in Hitachi. One of the reasons for my determination to join Hitachi was that there are many professionals with a wide variety of backgrounds, which gives me hope that I will also be able to become a professional of some kind.

Integration of Hitachi’s technologies in a single chip is bound to make quick identification of the causative agent of infections of the bloodstream possible

Yanagawa：The Biosystem Research Department is involved in the development of measurement technologies primarily for humans. They include genetic testing devices for life science research and blood testing devices for healthcare facilities. Four years ago, we started the development of methods for the quick identification of the causative bacteria of infections of the bloodstream to explore the possibility of applying genetic testing technology to some novel fields. Antimicrobial drugs are used for the treatment of bloodstream infections caused by bacteria. Death due to pneumonia was reduced drastically after penicillin, the worlds’ first antimicrobial agent, was put into practical use in 1942 during World War II. Thereafter, various antimicrobial drugs have been developed and put into use worldwide.

However, as antimicrobials began to be used repeatedly or over a long period of time, bacteria resistant to them emerged. Drug-resistant bacteria, which cannot be eradicated by antimicrobial drugs, are posing a serious problem. Infection by drug-resistant bacteria is difficult to treat with antimicrobial drugs. In the background of the recent COVID-19 pandemic, the threat of drug-resistant bacteria has been increasing, and it is called a “silent pandemic”. Ten million people are expected to be infected by drug-resistant bacteria and die annually in 2050.1 This figure is very large since the death toll from COVID-19 infection in 2021 is reported to be 3.6 million.

Shimizu：Concerning infections of the bloodstream, there are data indicating that the survival rate decreases by 7.6% with a one-hour delay of the administration of antimicrobials. The administration of antimicrobial drugs must be started as soon as possible. For the administration, appropriate antimicrobials must be selected according to the kind of bacteria, etc. Presently, however, it is difficult to immediately determine the bacteria causing the infection of the bloodstream, because they exist in the bloodstream in minute amounts and cannot be detected as they are. Testing is presently performed by increasing the bacteria by cultivation, which requires a total of 3 days or more. This is why doctors do “empiric treatments”.

In “empiric treatment”, the doctor prescribes antimicrobials by guessing the bacteria that are likely to be causing the infection from the age or underlying disease of the patient, probable circumstances of infection, history of the previous use of antimicrobials, organs suspected to be affected, and the state of epidemics in the facility or region. In this process, antimicrobials that are effective against a wide range of bacteria are selected to ensure that the bacteria causing the disease are covered. On the other hand, such antimicrobials that cover a wide range of bacteria exert adverse effects on bacteria other than the responsible bacteria, possibly resulting in the advent of drug-resistant strains. If the causative bacteria can be identified before the first administration of antimicrobials by rapid testing, it would become possible to prescribe the minimum possible antimicrobials according to the test results and, thus, to contribute to the control of the development of drug-resistant bacteria.

Yanagawa：Therefore, we decided to develop a method for the rapid identification of the causative bacteria of infections of the bloodstream using technologies that we have. We are proceeding with the development of a testing device that can quickly identify the causative bacteria in about 1 hour, which is a standard in “empiric treatment”.

As Ms. Shimizu has just explained, bacteria causing infections of the bloodstream are presently identified by cultivation, and it takes time, but our testing device does not need cultivation, since it amplifies the gene of the bacteria rather than the bacteria themselves. The device extracts DNA of bacteria from a blood sample in about 45 minutes, amplifies it, unravels it into single strands, and detects them (figure below). By putting a blood sample into a microfluidic device (also called flow chip, or µ-TAS) in which a variety of technologies are integrated, the processes necessary for the detection are performed on a single device.

Shimizu：We chose the methods for the extraction of bacterial DNA, amplification, preparation of single strands, and detection by comparing several methods for each process.
For DNA amplification, the sample fluid must be repeatedly heated and cooled 30-40 times.
Therefore, we adopted a method in which high-temperature and low-temperature areas are created using two heaters on the device, and the sample solution is shuttled between them. By this method, we could rapidly change the temperature of the sample solution and complete the DNA amplification reaction in about 8 minutes, while it usually takes several tens of minutes.
In amplified DNA, two strands are stuck together (double-stranded), but it must be divided into single strands to facilitate detection. We selected the enzymatic digestion method, which is efficient with a short reaction time, as the method for the preparation of single-stranded DNA.

DNA is detected using a bead array system. Single-stranded DNA of the bacteria to be detected (probe DNA) is attached in advance to beads for gene detection). If bacterial DNA, extracted from the blood sample amplified, and single-stranded, binds to the probe DNA, the fluorescent tag at the tip of the bacterial DNA glows, indicating that the bacteria are contained. In a bead array in which beads for gene detection were arranged, the occurrence of turbulent flow was observed around each bead, and the binding time of single-stranded DNA could be shortened. In the experiment, the time needed for amplification and detection could be reduced to 14 minutes if 100 copies of bacterial DNA could be extracted.

Yanagawa：Until we came to this point, we had many problems such as the loss of precious DNA extracted from bacteria as they were adsorbed on the wall of the microfluidic device and poor fluid transport. In the field of electronic circuits, which is my specialty, simulation technology is established, and the device often operates as designed, but the story is different in the field of bioscience working with biological samples such as DNA. Since there are many specialists in bioscience among the colleagues and senior members of the laboratory, we consulted with such people when we had difficulties and overcame problems together.

Shimizu：I am used to experiments of bioscience, but I had no experience in designing microfluidic device before. Now, I have become able to do rough designing using computer-assisted designing (CAD). I can ask questions to colleagues familiar with CAD or ask Mr. Yanagawa for help in parts involving electricity. Since there are only experts in the same field in university laboratories, the designing of a new device, as in this is a job, is special to Hitachi’s laboratory.

Yanagawa：I hope to become able to test about 40 species of bacteria. As there are global needs for the identification of the causative bacteria of infections of the bloodstream, competition has already begun. We are now hearing the opinions of doctors and laboratory technicians treating infections of the bloodstream about whether the device is clinically usable. Since the prescription of antimicrobials is important in clinical situations, and, in addition, the mortality rate increases on an hour-by-hour basis, we must avoid situations in which bacteria exist but are not detected (low sensitivity), or bacteria are detected but do not exist (low specificity) as much as possible. It is also important to design a testing device small enough to be installed in the clinical laboratory. I first aim to complete this device and then to improve the accuracy to a clinically applicable level.

¹ Citation: JIMO’ NEILL et al., Tackling Drug-resistant Infections Globally (2016)