崔 龍洙

Abstract In the ongoing COVID-19 pandemic, rapid and sensitive diagnosis of viral infection is a critical deterrent to the spread of SARS-CoV-2. To this end, we developed an automated amplification-free digital RNA detection platform using CRISPR-Cas13a and microchamber device (opn-SATORI), which automatically completes a detection process from sample mixing to RNA quantification in clinical specimens within ~9 min. Using the optimal Cas13a enzyme and magnetic beads technology, opn-SATORI detected SARS-CoV-2 genomic RNA with a LoD of < 6.5 aM (3.9 copies μL⁻¹), comparable to RT-qPCR. Additionally, opn-SATORI discriminated between SARS-CoV-2 variants of concern, including alpha, delta, and omicron, with 98% accuracy. Thus, opn-SATORI can serve as a rapid and convenient diagnostic platform for identifying several types of viral infections.

Non-menstrual toxic shock syndrome (non-mTSS) is a life-threatening disease caused by <italic>Staphylococcus aureus</italic> strains producing superantigens, such as staphylococcal enterotoxins A, B, C, and toxic shock syndrome toxin-1 (TSST-1). However, little is known about why the TSS cases are rare, although <italic>S. aureus</italic> strains frequently carry a <italic>tst</italic> gene, which encodes TSST-1. To answer this question, the amount of TSST-1 produced by 541 clinical isolates was measured in both the presence and absence of serum supplementation to growth media. Then a set of <italic>S. aureus</italic> strains with similar genetic backgrounds isolated from patients presenting with non-mTSS and those with clinical manifestations other than non-mTSS was compared for their TSST-1 inducibility by human serum, and their whole-genome sequences were determined. Subsequently, the association of mutations identified in the <italic>tst</italic> promoter of non-mTSS strains with TSST-1 inducibility by human serum was evaluated by constructing promoter replacement mutants and green fluorescent protein (GFP) reporter recombinants. Results showed that 39 out of 541 clinical isolates (7.2%), including strains isolated from non-mTSS patients, had enhanced production of TSST-1 in the presence of serum. TSST-1 inducibility by human serum was more clearly seen in non-mTSS strains of clonal complex (CC)-5. Moreover, the whole-genome sequence analysis identified a set of sequence variations at a putative SarA-binding site of the <italic>tst</italic> promoter. This sequence variation was proven to be partially responsible for the induction of TSST-1 production by human serum. We conclude that the onset of staphylococcal toxic shock syndrome caused by TSST-1-producing CC-5 strains seem at least partially initiated by serum induction of TSST-1, which is regulated by the mutation of putative SarA-binding site at the <italic>tst</italic> promoter.

<italic>Staphylococcus</italic> spp. colonize commensally on the human skin. Some commensal coagulase-negative staphylococci and <italic>Staphylococcus aureus</italic> are also involved in nosocomial infections. Bacteria were collected from skin healed from pressure injury (PI). After the collection time points, some patients suffered from recurrent PI (RPI). This study analyzed the characteristics of <italic>Staphylococcus</italic> spp. on healed skin before recurrence between healed skin that suffered from RPI within 6 weeks (RPI group) and healed skin that did not suffer within the duration (non-RPI group) by <italic>Staphylococcus</italic> spp.-specific sequencing. Of the seven patients in the RPI group, two were dominated by <italic>S. aureus</italic> and four by <italic>Staphylococcus caprae</italic>, coagulase-negative human commensal staphylococci in the RPI group. Using mouse models, both <italic>S. caprae</italic> and <italic>S. aureus</italic>, but not <italic>Staphylococcus epidermidis</italic>, colonized on skin healed from injury at significantly higher rates than normal skin. Although subcutaneous injection of <italic>S. caprae</italic> did not induce lesion formation, the bacterium exhibited high hemolytic activity on human red blood cells. Lesion formation by subcutaneous injection of <italic>S. aureus</italic> was significantly suppressed in the presence of <italic>S. caprae</italic>. The hemolytic activity of rabbit blood cells of <italic>S. aureus</italic> was suppressed by <italic>S. caprae</italic>, whereas the hemolytic activity of <italic>S. caprae</italic> was dramatically suppressed by <italic>S. aureus</italic>. Data indicated that each of the two <italic>Staphylococcus</italic> spp. suppresses the pathogenicity of the other and that the imbalance between the two is associated with RPI.

A high prevalence of extended-spectrum β-lactamase-producing Enterobacterales (ESBL-PE) may call for monitoring in geriatric long-term care facilities (g-LTCFs). We surveyed the distribution of ESBL-causative gene types and antimicrobial susceptibility in ESBL-PE strains from residents in g-LTCFs, and investigated the association between ESBL-causative gene types and antimicrobial susceptibility. First, we analyzed the types of ESBL-causative genes obtained from 141 ESBL-PE strains collected from the feces of residents in four Japanese g-LTCFs. Next, we determined the minimum inhibitory concentration values for alternative antimicrobial agents against ESBL-PE, including β-lactams and non-β-lactams. Escherichia coli accounted for 96% of the total ESBL-PE strains. Most strains (94%) contained blaCTX-M group genes. The genes most commonly underlying resistance were of the blaCTX-M-9 and blaCTX-M-1 groups. Little difference was found in the distribution of ESBL-causative genes among the facilities; however, antimicrobial susceptibility differed widely among the facilities. No specific difference was found between antimicrobial susceptibility and the number of ESBL-causative genes. Our data showed that ESBL-PEs were susceptible to some antimicrobial agents, but the susceptibility largely differed among facilities. These findings suggest that each g-LTCF may require specific treatment strategies based on their own antibiogram. Investigations into drug resistance should be performed in g-LTCFs as well as acute medical facilities.

INTRODUCTION: From 2018, IR Biotyper (IRBT; Bruker Daltonik GmbH, Germany) based on the Fourier transform infrared spectrophotometer has begun to be introduced as a new strain classification method in the field of clinical microbiological examination. We compared it with molecular epidemiology method to evaluate the usefulness of strain classification by IRBT. METHOD: Homology of strain classification with molecular epidemiology method (Multilocus Sequencing Typing; MLST and PCR-based ORF Typing; POT) for 48 strains of Pseudomonas aeruginosa with different detection times from multiple institutions to evaluate the accuracy of IRBT was compared. RESULTS: IRBT used "KBM" SCD agar medium for preculture and was classified into 12 types when classified by Cut-off value 0.181, 8 types by MLST, and 13 types by POT. In the Adjusted Wallace between IRBT and molecular epidemiology method, MLST was 0.458 (95% CI; 0.295 to 0.620) and POT was 0.330 (95% CI; 0.135 to 0.525), indicating a discrepancy in strain classification. CONCLUSION: No correlation was found between IRBT and the classification results by the molecular epidemiology method. In the molecular epidemiology method, strains are classified by matching only specific gene regions, but IRBT irradiates a sample with an infrared laser and classifies the strains according to the difference in absorption spectrum according to the molecular structure, so the measurement principle is different. When classifying strains by IRBT, it is desirable to grasp the clinical information of the detected strains and to target multiple strains isolated at the same facility at the same time.

<title>Abstract</title>Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are one of the factors which can contribute to limiting the development and evolution of antibiotic resistance in bacteria. There are three genomic loci of CRISPR-Cas in <italic>Enterococcus faecalis</italic>. In this study, we aimed to assess correlation of the CRISPR-Cas system distribution with the acquisition of antibiotic resistance among <italic>E. faecalis</italic> isolates. A total of 151 isolates of <italic>E. faecalis</italic> were collected from urinary tract infections (UTI) and dental-root canal (DRC). All isolates were screened for phenotypic antibiotic resistance. In addition, antibiotic resistance genes and CRISPR loci were screened by using polymerase chain reaction. Genomic background of the isolates was identified by random amplified polymorphic DNA (RAPD)-PCR. The number of multidrug-resistant <italic>E. faecalis</italic> strains were higher in UTI isolates than in DRC isolates. RAPD-PCR confirmed that genomic background was diverse in UTI and DRC isolates used in this study. CRISPR loci were highly accumulated in gentamycin-, teicoplanin-, erythromycin-, and tetracycline-susceptible strains. In concordance with drug susceptibility, smaller number of CRISPR loci were identified in <italic>vanA</italic>, <italic>tetM</italic>, <italic>ermB</italic>, <italic>aac6’-aph(2”)</italic>, <italic>aadE</italic>, and <italic>ant(6)</italic> positive strains. These data indicate a negative correlation between CRISPR-<italic>cas</italic> loci and antibiotic resistance, as well as, carriage of antibiotic resistant genes in both of UTI and DRC isolates.

Previous studies indicated residents in geriatric long-term care facilities (LTCFs) had much higher prevalence of extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E) carriage than the general population. Most ESBL-E carriers are asymptomatic. The study tested the hypothesis that residents with ESBL-E carriage may accumulate inside geriatric LTCFs through potential cross-transmission after exposure to residents with prolonged ESBL-E carriage. 260 residents from four Japanese LTCFs underwent ESBL-E testing of fecal specimens and were divided into two cohorts: Cohort 1,75 patients with ≥ 2 months residence at study onset; Cohort 2, 185 patients with < 2 months residence at study onset or new admission during the study period. Three analyses were performed: (1) ESBL-E carriage statuses in Cohort 1 and Cohort 2; (2) changes in ESBL-E carriage statuses 3-12 months after the first testing and ≥ 12 months after the second testing; and (3) lengths of positive ESBL-E carriage statuses. Compared with the residents in Cohort 1, a significantly larger proportion of residents in Cohort 2 were positive for ESBL-E carriage (28.0% in Cohort 1 vs 40.0% in Cohort 2). In the subsequent testing results, 18.3% of residents who were negative in the first testing showed positive conversion to ESBL-E carriage in the second testing, while no patients who were negative in the second testing showed positive conversion in the third testing. The maximum length of ESBL-E carriage was 17 months. The findings indicated that some residents acquired ESBL-E through potential cross-transmission inside the LTCFs after short-term residence. However, no residents showed positive conversion after long-term residence, which indicates that residents with ESBL-E carriage may not accumulate inside LTCFs. Practical infection control and prevention measures could improve the ESBL-E prevalence in geriatric LTCFs.

<title>Abstract</title><sec> <title>Purpose</title> We aimed to investigate whether low-intensity continuous and pulsed wave ultrasound (US) irradiation can inhibit the formation of <italic>Staphylococcus epidermidis</italic> biofilms, for potential application in the treatment of catheter-related bloodstream infections (CRBSI). </sec><sec> <title>Methods</title> <italic>S. epidermidis</italic> biofilms that formed on the bottom surfaces of 6-well plates were irradiated on the bottom surface using the sound cell incubator system for different intervals of time. </sec><sec> <title>Results</title> US irradiation with continuous waves for 24 h notably inhibited biofilm formation (<italic>p</italic> &lt; 0.01), but the same US irradiation for 12 h had no remarkable effect. Further, double US irradiation with pulsed waves for 20 min inhibited biofilm formation by 33.6%, nearly two-fold more than single US irradiation, which reduced it by 17.9%. </sec><sec> <title>Conclusion</title> US irradiation of a lower intensity (<italic>I</italic>_SATA = 6–29 mW/cm²) than used in a previous study and lower than recommended by the Food and Drug Administration shows potential for preventing CRBSI caused by bacterial biofilms. </sec>

The bacteriophage (or phage for short) has been used as an antibacterial agent for over a century but was abandoned in most countries after the discovery and broad use of antibiotics. The worldwide emergence and high prevalence of antimicrobial-resistant (AMR) bacteria have led to a revival of interest in the long-forgotten antibacterial therapy with phages (phage therapy) as an alternative approach to combatting AMR bacteria. The rapid progress recently made in molecular biology and genetic engineering has accelerated the generation of phage-related products with superior therapeutic potentials against bacterial infection. Nowadays, phage-based technology has been developed for many purposes, including those beyond the framework of antibacterial treatment, such as to suppress viruses by phages, gene therapy, vaccine development, etc. Here, we highlighted the current progress in phage engineering technology and its application in modern medicine.

We report the complete genome sequence of <named-content content-type="genus-species">Mycobacterium heckeshornense</named-content> strain JMUB5695, which was isolated from necrotizing granulomatous lesions in a lung cancer patient. The complete genome consists of a 4,865,109-bp chromosome with a GC content of 65.9% and contains no plasmids.

Influenza outbreaks at geriatric long-term care facilities (g-LTCFs) can be deadly and their prevention is important. However, the factors influencing disease transmission in g-LTCFs remain controversial. In this descriptive study, we tried to identify the potential risk factors influencing influenza outbreaks that occurred in different influenza seasons within a single g-LTCF with 100 residents in Gunma Prefecture. We reviewed the detailed facility records for all influenza cases in both residents and staff between January 2012 and June 2020. Facility preventive measures were also reviewed. We found that community meals may have been a potential source of transmission leading to the outbreaks. When influenza infection is noted, implementation of strict preventive measures and restriction of meal provision to resident rooms may help to prevent disease transmission and the development of an outbreak. Such measures may also be useful to prevent the transmission of other serious droplet-borne diseases within g-LTCFs.

Differences in the gut microbial content of Lutzomyia (Lu.) ayacuchensis, a primary vector of Andean-type cutaneous leishmaniasis in Ecuador and Peru, may influence the susceptibility of these sand flies to infection by Leishmania. As a first step toward addressing this hypothesis, a comparative analysis of bacterial and fungal compositions from Lu. ayacuchensis populations with differential susceptibilities to Leishmania was performed. Bacterial 16S rRNA gene amplification and Illumina MiSeq sequencing approaches were used to characterize the bacterial composition in wild-caught populations from the Andean areas of Ecuador and southern Peru at which the sand fly species transmit Leishmania (Leishmania) mexicana and Leishmania (Viannia) peruviana, respectively, and a population from the northern Peruvian Andes at which the transmission of Leishmania by Lu. ayacuchensis has not been reported. In the present study, 59 genera were identified, 21 of which were widely identified and comprised more than 95% of all bacteria. Of the 21 dominant bacterial genera identified in the sand flies collected, 10 genera had never been detected in field sand flies. The Ecuador and southern Peru populations each comprised individuals of particular genera, while overlap was clearly observed between microbes isolated from different sites, such as the number of soil organisms. Similarly, Corynebacterium and Micrococcus were slightly more dominant bacterial genera in the southern Peru population, while Ochrobactrum was the most frequently isolated from other populations. On the other hand, fungi were only found in the southern Peru population and dominated by the Papiliotrema genus. These results suggest that variation in the insect gut microbiota may be elucidated by the ecological diversity of sand flies in Peru and Ecuador, which may influence susceptibility to Leishmania infection. The present study provides key insights for understanding the role of the microbiota during the course of L. (L.) mexicana and L. (V.) peruviana infections in this important vector.

The emergence of antimicrobial-resistant bacteria is an increasingly serious threat to global health, necessitating the development of innovative antimicrobials. Here we report the development of a series of CRISPR-Cas13a-based antibacterial nucleocapsids, termed CapsidCas13a(s), capable of sequence-specific killing of carbapenem-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus by recognizing corresponding antimicrobial resistance genes. CapsidCas13a constructs are generated by packaging programmed CRISPR-Cas13a into a bacteriophage capsid to target antimicrobial resistance genes. Contrary to Cas9-based antimicrobials that lack bacterial killing capacity when the target genes are located on a plasmid, the CapsidCas13a(s) exhibit strong bacterial killing activities upon recognizing target genes regardless of their location. Moreover, we also demonstrate that the CapsidCas13a(s) can be applied to detect bacterial genes through gene-specific depletion of bacteria without employing nucleic acid manipulation and optical visualization devices. Our data underscore the potential of CapsidCas13a(s) as both therapeutic agents against antimicrobial-resistant bacteria and nonchemical agents for detection of bacterial genes.

The association of Panton-Valentine leukocidin (PVL) toxin with necrotizing soft tissue infection (NSTI) caused by <named-content content-type="genus-species">Staphylococcus aureus</named-content> remains controversial. Here, we report the complete genome sequence of the PVL-negative <named-content content-type="genus-species">S. aureus</named-content> strain JMUB1273, isolated from a patient with pervasive NSTI.

[This corrects the article DOI: 10.14252/foodsafetyfscj.2018003.].

Severe community-acquired pneumonia (CAP) caused by methicillin-resistant Staphylococcus aureus (MRSA) is relatively rare and is usually associated with rapid progression to death. Here, we report the complete genome sequence of the MRSA strain JMUB3031, which was isolated from a patient with fatal CAP.

BACKGROUND: Staphylococcus caprae is an animal-associated bacterium regarded as part of goats' microflora. Recently, S. caprae has been reported to cause human nosocomial infections such as bacteremia and bone and joint infections. However, the mechanisms responsible for the development of nosocomial infections remain largely unknown. Moreover, the complete genome sequence of S. caprae has not been determined. RESULTS: We determined the complete genome sequences of three methicillin-resistant S. caprae strains isolated from humans and compared these sequences with the genomes of S. epidermidis and S. capitis, both of which are closely related to S. caprae and are inhabitants of human skin capable of causing opportunistic infections. The genomes showed that S. caprae JMUB145, JMUB590, and JMUB898 strains contained circular chromosomes of 2,618,380, 2,629,173, and 2,598,513 bp, respectively. JMUB145 carried type V SCCmec, while JMUB590 and JMUB898 had type IVa SCCmec. A genome-wide phylogenetic SNP tree constructed using 83 complete genome sequences of 24 Staphylococcus species and 2 S. caprae draft genome sequences confirmed that S. caprae is most closely related to S. epidermidis and S. capitis. Comparative complete genome analysis of eight S. epidermidis, three S. capitis and three S. caprae strains revealed that they shared similar virulence factors represented by biofilm formation genes. These factors include wall teichoic acid synthesis genes, poly-gamma-DL-glutamic acid capsule synthesis genes, and other genes encoding nonproteinaceous adhesins. The 17 proteinases/adhesins and extracellular proteins known to be associated with biofilm formation in S. epidermidis were also conserved in these three species, and their biofilm formation could be detected in vitro. Moreover, two virulence-associated gene clusters, the type VII secretion system and capsular polysaccharide biosynthesis gene clusters, identified in S. aureus were present in S. caprae but not in S. epidermidis and S. capitis genomes. CONCLUSION: The complete genome sequences of three methicillin-resistant S. caprae isolates from humans were determined for the first time. Comparative genome analysis revealed that S. caprae is closely related to S. epidermidis and S. capitis at the species level, especially in the ability to form biofilms, which may lead to increased virulence during the development of S. caprae infections.

Electroporation is a common technique necessary for genomic manipulation of Staphylococci. However, because this technique has too low efficiency to be applied to some Staphylococcal species and strains, especially to coagulase-negative Staphylococcus (CNS) isolates, basic researches on these clinically important Staphylococci are limited. Here we report on the optimization of electroporation parameters and conditions as well as on the generation of a universal protocol that can be efficiently applicable to both CNS and Coagulase-positive Staphylococci (CPS). This protocol could generate transformants of clinical Staphylococcus epidermidis isolate, with an efficiency of up to 1400 CFU/μg of plasmid DNA. Transformants of 12 other clinically important Staphylococcal species, including CNS and CPS, were also generated with this protocol. To our knowledge, this is the first report on successful electroporation in nine these Staphylococcal species.

BACKGROUND: Increasing evidence has indicated that Staphylococcus aureus pneumonia complicated with influenza virus infection is often fatal. In these cases, disease severity is typically determined by susceptibility to antimicrobial agents and the presence of high-virulence factors that are produced by Staphylococcus aureus, such as Panton-Valentine leukocidin (PVL). CASE REPORT: We describe a rare case of fatal community-acquired pneumonia caused by methicillin-sensitive Staphylococcus aureus (MSSA), which did not secrete major high-virulence factors and coexisted with influenza type B infection. The 32-year-old previously healthy male patient presented with dyspnea, high fever, and cough. His roommate had been diagnosed with influenza B virus infection 3 days earlier. Gram-positive clusters of cocci were detected in the patient's sputum; therefore, he was diagnosed with severe pneumonia and septic shock, and was admitted to the intensive care unit. Despite intensive antibiotic and antiviral treatment, he died of multiple organ failure 5 days after admission. His blood culture from the admission was positive for MSSA, and further analysis revealed that the strain was negative for major high-virulence factors, including PVL and enterotoxins, although influenza B virus RNA was detected by PCR. CONCLUSIONS: Physicians should pay special attention to patients with pneumonia following influenza and Staphylococcus aureus infection, as it may be fatal, even if the Staphylococcus aureus strain is PVL-negative and sensitive to antimicrobial agents.