In a pandemic crisis where we must face an unknown infectious disease, it is necessary to accurately and rapidly assess the immune status of individuals or groups in real-time against the causative microorganisms. Nevertheless, in the current COVID-19 pandemic, SARS-CoV-2 antibody testing requires serum or plasma collected by venipuncture. However, this sample collection poses several logistical restrictions, such as the requirements of venipuncture by trained phlebotomists with direct contact, immediate storage in a refrigerator or freezer after collection, and cold chain transport to maintain the integrity of the biospecimen. In contrast, dried blood spot (DBS) sampling is simple and inexpensive; DBS samples can be self-collected and remain stable at ambient temperature until sent by postal mail to a laboratory for processing. Moreover, DBS sampling has the potential to provide a widely available solution for assessing and tracking herd immunity in various populations, including those in low- and middle-income countries, without geographic limitations. The present study is the first to demonstrate the correlations between neutralizing antibody responses not only against the wild-type strain but also BA.5 and XBB.1.5 in the DBS eluate and paired sera. In addition, neutralizing antibodies have been shown to remain stable in collected DBS transported at HT (40°C) for one month, following one-month storage at room temperature. However, in our experiments, the correlations and stability over time and temperature varied depending on the type of filtered papers used, with filter paper no. 545 being the most suitable for DBS cards in the high-throughput chemiluminescent reduction neutralizing test (htCRNT) assay. Evaluating neutralizing antibodies using DBS eluates could be applied to other pathogens and in future pandemics, enabling reliable and affordable seroepidemiological surveillance, even in remote areas and low-income countries.

Methicillin-resistant Staphylococcus aureus (MRSA) is the most detected drug-resistant organism in Japan. MRSA is sometimes one of the normal flora on the skin and in the nasal cavity. It can cause severe infections, such as pneumonia and bloodstream infections in immunocompromised patients. In bloodstream infections, MRSA is the most detected drug-resistant bacteria. However, in recent years, its detection rate has declined due to infection control and appropriate use of antimicrobial agents. In the past ten years, however, community-acquired MRSA (CA-MRSA) has emerged as a global problem, and USA-300, which produces panton valentine leukocidin (PVL) and causes severe conditions as necrotizing pneumonia, has been reported in North America. In Japan, however, the proportion of PVL-positive CA-MRSA is low, and CA-MRSA may be sensitive to antibacterial agents other than beta-lactams. MRSA bloodstream infections have a high mortality rate, and some studies have shown the benefit of combination therapy with anti-MRSA agents and other agents.

Therefore, combination therapy of anti-MRSA agents with sensitive antimicrobial agents may be a treatment option for bloodstream infections caused by CA-MRSA. However, from the viewpoint of the appropriate use of antimicrobial agents, it is desirable to confirm that the drugs used in combination with anti-MRSA agents are sensitive to the detected MRSA before use. At present, the use of mass spectrometry (MALDI-TOF/MS) and fully automated genetic testing has made it possible to identify MRSA more rapidly than the conventional method of culture testing. However, there is also the problem that it is not possible to detect drug resistance genes comprehensively. Recently, with the advent of next-generation sequencing, whole-genome sequencing (WGS) can be performed more quickly and easily than before. WGS data can be used not only for the comprehensive detection of drug resistance genes but also for identifying genotypes and pathogenic factors, making it a useful microbiological test. However, there are many issues to be solved before implementing WGS as a routine microbiological test. Therefore, it is important to conduct various verifications, such as detection of drug-resistant genes by WGS and comparison with drug susceptibility tests.

The recent COVID-19 pandemic has raised serious concerns regarding disease and human health. In terms of bacterial infection, antimicrobial resistance is a global issue that threatens to worsen infections. Antimicrobial agents used to treat bacterial infections have been evaluated with artificial media such as Mueller–Hinton medium. However, several reports show that data obtained from artificial media do not always correspond to reactions in vivo, suggesting that effective compounds may have been overlooked by conventional screening methods. If compounds are evaluated under conditions similar to those inside the human body, assessments of effective compounds may be made more accurate. Experimental animals have been employed as a method to provide in vivo information, but animal models may not translate across species and could, furthermore, create ethical issues around their use. 3D organotypic tissue models have attracted attention in this context. As these models are composed of human culture cell lines, problems surrounding species compatibility and ethics can be avoided. In addition, the state of the model is closer to that of the human body than either monolayer cell culture or artificial media. The 3D tissue model can thus be used to reproduce infectious diseases in vitro, as well as evaluate therapeutic drugs under environmental conditions similar to those found in the human body. This review summarizes the characteristics of the 3D tissue model and its construction method and discusses current infection models for respiratory tract infections.

The spread of carbapenem-resistant Enterobacterales(CRE) and the high mortality rate of CRE infections are serious problems in the antimicrobial therapy. CRE is a multidrug-resistant that has acquired resistance to various antibacterial, and its resistance genes and antibacterial susceptibility are regional. Antibacterial combination therapy is used to treat CRE infections, but there are concerns about adverse reactions to colistin (CL) and tigecycline (TGC), which are expected to be effective. In recent years, new antibacterial that are effective against CRE have been developed, but it will take some time before they are approved in Japan. For the time being, CRE infections will have to treat by existing antibacterial, and the challenge is how to enhance the ineffective ones to make them effective. Therefore, considering combination therapies that do not use CL and TGC is important for establishing treatment guidelines for future CRE infections.

Purpose: It is necessity of new rapid method for detecting microorganisms in bloodstream infection. Especially, patients with febrile-neutropenia tends to be poor prognosis. We focused on the new sequencing system, MinION. The new third-generation sequencing platform MinION is an attractive maintenance-free and disposable portable tool that can perform long reads and real-time sequencing. In this study, we validated this technology for the identification of pathogens from positive blood culture (BC) bottles.

Methodology: A total of 38 positive BC bottles collected from the patients of bloodstream infection, among which 18 isolates of Gram-negative (GN) bacteria and 19 isolates of Grampositive (GP) bacteria were identified using16S rRNA sequencing, were enrolled in this study. One case detected fungus from blood culture bottles. DNA was extracted from each aliquot using an extraction protocol that combined glass bead-beating and chemical lysis. Up to 200 ng of each purified DNA sample was processed for library preparation and whole genome sequencing was performed on up to 12 samples through a single Minion Flow Cell.

Results: All GN bacteria identified on the basis of the most frequent classified reads by MinION sequencing for 30 min using WIMP via EPI2ME were consistent with those identified by 16S rRNA sequencing. On the other hand, the results of identification by 16S rRNA sequencing and those by MinION agreed in 12 of 19 GP bacteria specimens. The ARMA analysis was able to detect extended spectrum β-lactamase (ESBL)-associated genes among various antimicrobial resistance-related genes.

Conclusion: We demonstrated the potential of the MinION sequencer for the identification of GN bacteria from positive BC bottles and its confirmation of an ESBL phenotype. This innovative sequence technology and application could be a breakthrough technology for the diagnosis of infectious diseases.

In recent years, the increase in the incidence of hypermucoviscous Klebsiella pneumoniae (hvKP) infections has become a clinical concern worldwide. The high pathogenicity of hvKP is in part attributed to the overproduction of capsular polysaccharide (CPS), which is achieved by the action of a positive regulator of capsular polysaccharide synthesis genes, named rmpA. There have been many studies on the mechanism of hvKP virulence, but no study has focused on the development of anti-virulence therapies. We tried to identify antimicrobial drugs that can suppress the hypermucoviscosity of hvKP. As a result, we found that rifampicin (RFP) has a strong anti-mucoviscous activity against hvKP. RFP treatment caused a drastic reduction in the thickness of the CPS layer around hvKP cells and suppressed transcript levels of capsule-related genes including rmpA. Our data suggest that RFP may be useful as a potential anti-virulence agent for intractable infections caused by hvKP.

Therapeutic hypothermia is the recommended regimen for adult subjects after cardiac arrest and in neonates with hypoxic ischemic encephalopathy. Remedies are needed to negate side effects or to provide sedation during hypothermia. However, changes in the pharmacokinetics of drugs have been reported under hypothermic conditions. The mechanisms responsible for changes in the pharmacokinetics of drugs under hypothermic conditions have not been clarified fully. To optimize the medication during therapeutic hypothermia, we need to clarify the factor affecting drug disposition. The factor affecting drug disposition under hypothermia is described in this review.

Influenza A and B viruses possess an enzyme “sialidase” that cleavages terminal sialic acid from glycochains. These viral sialidase proteins are highly expressed on the virus infected cells. We developed sialidase imaging probe “BTP3-Neu5Ac” that enables histochemical fluorescence staining of sialidase activity. BTP3-Neu5Ac was able to perform speedy and easy fluorescence imaging of these virus infected cells, with no needs of specific antibody and cell fixation. In addition, combination use of anti-influenza drugs (sialidase inhibitors) and BTP3-Neu5Ac resulted in selective fluorescence imaging for detection and high-efficiency isolation of drugresistant virus. Fluorescence imaging of drug-resistant virus will be a powerful method for study of the drug-resistance mechanism, for monitoring of drug-resistant viruses. A novel tool for fluorescence imaging of viral sialidase activity is described in this review.

Dissemination of carbapenem-resistant Enterobacteriaceae (CRE) poses a considerable threat to public health. Infections caused by CRE have limited treatment options and have been associated with high mortality rates. This resistance is largely the consequence of acquisition of carbapenemase genes. The genotypes in the geographical areas were initially different, as the Japanese epidemic was related to the IMP type (Class B), whereas the US epidemic was related to the KPC type( Class A). Thus, clinical detection of carbapenemase producers remains difficult based on each genotypes has specific screening method. CRE has many problems: some carbapenemase producers were susceptible to the carbapenems and some CRE were not producing carbapenemase which is associated with porin loss or efflux pomp. This review describes the current situation of CRE. It would facilitate accurate detection of CRE and approaches to prevention and treatment.

The balance of “Host-Pathogen-Antimicrobials” is crucial for the establishment of infectious diseases. Recently the ineffective cases, even though the appropriate antibiotics use, have been increasing since the several ineludible problems are rising, such as varied patient’s background and the epidemic of the drug resistant pathogens, etc. Despite of the medical progression and the development of novel antimicrobials, the mortality of pneumonia has increased gradually and been the third cause of death in Japan. The conventional treatment depended on only bactericidal effect of antimicrobials faces a limit and the alternative strategies are required to overcome the current situation. This review addresses the potency of non-antibiotic antimicrobial agents as an alternative therapeutic strategy, especially focused on the activation of the innate host immunity induced by the macrolides and toll-like receptor agonist.