Molecular characteristics of methicillin-resistant Staphylococcus aureus isolated from skin and soft tissue infections collected in the Japanese nationwide surveillance

Skin and soft tissue infections (SSTI) are a common infection among both outpatients and inpatients. The most frequently isolated bacterium in SSTI was Staphylococcus aureus, a quarter of which was methicillin-resistant S. aureus (MRSA). In this study, to investigate molecular epidemiology of the 141 MRSA strains collected in the Japanese nationwide surveillance, we performed multiplex real-time polymerase chain reaction to detect staphylococcal cassette chromosome mec (SCCmec) type and virulence genes. The percentage of SCCmec types I, II, III and IV was 1.4%, 52.5%, 5.7% and 40.4%, respectively. According to the SCCmec type, we classified the strains into health-care-associated (HA)-MRSA (n = 84) and community-associated (CA)-MRSA (n = 57). Among the virulence genes, the percentage of enterotoxin C gene-positive strains was significantly higher in CA-MRSA than in HA-MRSA. No significant differences were detected between the two groups in terms of antibiotic susceptibility and patients’ background information, classification of SSTI or symptoms of SSTI

Molecular epidemiology of Clostridioides difficile and risk factors for the detection of toxin gene-positive strains

In this study, we investigated all Clostridioides difficile strains isolated from stool samples in Nagasaki University Hospital between January 2012 and December 2014. Toxin genes (tcdA, tcdB and cdtA/cdtB) were analyzed for multiplex PCR in a total of 213 strains. In the toxin gene-positive strain, PCR ribotyping was conducted using capillary gel electrophoresis-based PCR and the Webribo database. Patients’ backgrounds were analyzed by departments, disorders, antimicrobials, and clinical dates. The positive rates of tcdA, tcdB, and cdtA/cdtB genes were 62.9%, 63.4%, and 2.8%, respectively. The most frequent PCR ribotype was 047 (14.1%), followed by 014/0 (11.1%) and 002/0 (8.2%). In univariate analysis, the risk factors for the detection of toxin gene-positive strains in patients were older age (p = 0.0036), over ≥ 65 years old (p = 0.0175), the patients hospitalized at Department of Digestive Surgery (P = 0.0059), higher CRP level (P = 0.0395), and lower albumin level (p = 0.0014). In the multivariate analysis, the risk factor for detection of toxin gene-positive strains was the patients hospitalized at Department of Digestive Surgery (OR; 4.62, 95% CI; 1.18–18.0, p = 0.0274). In this study, the percentage of toxin gene-positive and cdtA/cdtB gene-positive strains was almost the same as that reported in previous studies, but the ribotype was different. In addition, we revealed that the risk factor associated with the detection of toxin gene-positive strains was the patients hospitalized at Department of digestive surgery

Detection of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae using the MALDI Biotyper Selective Testing of Antibiotic Resistance?β-Lactamase (MBT STAR-BL) assay

The MALDI Biotyper Selective Testing of Antibiotic Resistance?β-Lactamase (MBT STAR-BL) assay, which analyzes bacterial induced hydrolysis of cefotaxime using MALDI-TOF MS, correctly identified 100.0% of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae as positive and 94.7% of non-ESBL producers as negative in 80 strains tested

The surveillance of colistin resistance and mobilized colistin resistance genes in multidrug-resistant Enterobacteriaceae isolated in Japan

Background: The plasmid-mediated bacterial colistin-resistant gene, mcr, is of global concern in clini-cal healthcare. However, there are few reports of surveillance for mcr in Japan. The aim of this study was to assess the prevalence of colistin resistance by identifying nine mcr genes in extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae and carbapenem-resistant Enterobacteriaceae (CRE) isolates in Japan.Methods: A total of 273 ESBL and CRE clinical isolates were collected from patients in five tertiary hospi-tals from August 2016 to March 2017. Minimum inhibitory concentration (MIC) of colistin was measured using the microdilution method. Polymerase chain reaction (PCR) was performed to detect mcr-1 to mcr-9 genes in all strains. Whole-genome sequencing (WGS) analysis was conducted for any mcr-genes identi-fied that had not been previously reported in patients from Japan.Results: The rate of colistin resistance was 7.7% in all strains, with a higher rate in the CRE strains than in the ESBL-producing strains (20.4% versus 1.1%). The mcr-5 and mcr-9 gene were detected in one ESBL-producing Escherichia coli strain (1/273, 0.37%) and three CRE strains (3/273, 1.1%), respectively. As theESBL-producing E. coli strain was the first clinical strain with mcr-5 in Japan, WGS analysis was performed for the strain. The sequence type of the mcr-5-positive strain was ST1642 and it carried two distinct plasmids, ESBL gene-carrying pN-ES-6-1, and mcr-5.1-carrying pN-ES-6-2.Conclusions: The results of this study showed that the frequency of colistin resistance and mcr-positive strains is not high in Japan. As the MIC for colistin was low in the mcr-5.1 and mcr-9 gene-positive strain, continuous monitoring of mcr genes is necessary

成人T細胞白血病/リンパ腫のクローン構成を評価する新規方法の開発

長崎大学学位論文 [学位記番号]博（医歯薬）甲第1308号 [学位授与年月日]令和3年3月3

Antimicrobial and immunomodulatory effects of tedizolid against methicillin-resistant Staphylococcus aureus in a murine model of hematogenous pulmonary infection

Tedizolid (TZD) is a second-generation oxazolidinone and demonstrates potent in-vitro activity against multidrug-resistant Gram-positive bacteria. Phase III studies in patients with acute bacterial skin and skin structure infections (ABSSSI) have demonstrated the non-inferiority of TZD to linezolid (LZD). However, there are only a few studies that show the effect of TZD in pulmonary infections. In this study, we investigated the effect of TZD in a murine model of hematogenous pulmonary infection caused by methicillin-resistant Staphylococcus aureus (MRSA). The mice were treated either twice daily with saline (control), 25 mg/kg of vancomycin (low-VAN), 110 mg/kg of vancomycin (high-VAN), 120 mg/kg of LZD or once daily with 20 mg/kg of TZD. As compared to the control, the low- and high-VAN treatment groups, LZD and TZD significantly improved the survival rate, reduced the bacterial count in the lungs. Furthermore, TZD decreased the area of central bacterial colony zone (CBCZ) at 36 h post-inoculation, compared with the control. In addition, we investigated the immunomodulatory effect of TZD by evaluating the plasma concentrations of the inflammatory cytokines. Although there were no significant differences in the bacterial count in the lungs amongst the drugs at 26 h post-inoculation, TZD and LZD significantly improved the plasma concentrations of TNF-alpha, IL-6 and MIP-2, in comparison with the control. In this study, both TZD and LZD demonstrated antimicrobial and immunomodulatory efficacy in a murine model of hematogenous pulmonary infection caused by MRSA

Immunomodulatory Effect of Linezolid on Methicillin-Resistant Staphylococcus aureus Supernatant-Induced MUC5AC Overexpression in Human Airway Epithelial Cells

Linezolid is the first member of the oxazolidinones and is active against drug-resistant Gram-positive pathogens, such as methi-cillin- resistant Staphylococcus aureus (MRSA). Additionally, linezolid shows an immunomodulatory effect, such as inhibition of inflammatory cytokine production. In this study, we examined the effect of linezolid on MRSA-induced MUC5AC overexpression in airway epithelial cells. In this study, an MRSA supernatant was used to avoid the direct effect of linezolid on MRSA. MUC5AC protein production was significantly increased with a 40-fold dilution of MRSA supernatant. At the mRNA level, MUC5AC gene expression was significantly increased 6 and 9 h after stimulation. In an inhibition study, linezolid significantly reduced MRSA-induced MUC5AC protein and mRNA overexpression at concentrations of 5 and 20 μg/ml, which were the same as the trough and peak concentrations in human epithelial lining fluid. In an analysis of cell signaling, among the mitogen-activated protein kinase inhibitors, only the extracellular signal-regulated protein kinase 1/2 (ERK1/2) inhibitor reduced the MUC5AC protein production to the same level as that of the control; on Western blot analysis, only ERK1/2 was phosphorylated by the MRSA supernatant. In addition, the ERK1/2 phosphorylation was inhibited by linezolid. MUC5AC and MUC5B are the major barrier that traps inhaled microbial organisms, particulates, and foreign irritants. However, in patients with chronic respiratory diseases, pathogen-induced MUC5AC overexpression causes many problems, and control of the overexpression is important. Thus, this study revealed that linezolid showed a direct immunomodulatory effect in airway epithelial cells

Efficacy and pharmacokinetics of ME1100, a novel optimized formulation of arbekacin for inhalation, compared with amikacin in a murine model of ventilator-associated pneumonia caused by Pseudomonas aeruginosa

Background: Arbekacin is an aminoglycoside that shows strong antimicrobial activity against Gram-positive bacteria, including MRSA, as well as Pseudomonas aeruginosa. The therapeutic effectiveness of arbekacin is directly related to Cmax at the infection site. To maximize drug delivery to the respiratory tract and minimize the systemic toxicity, arbekacin optimized for inhalation, ME1100, is under development. In this study, we investigated the efficacy and pharmacokinetics of ME1100 in a murine model of ventilator-associated pneumonia caused by P. aeruginosa by using a customized investigational nebulizer system. Methods: The mice were treated for 5 min, once daily, with placebo, 3, 10 or 30 mg/mL ME1100 or 30 mg/mL amikacin. Results: In the survival study, the survival rate was significantly improved in the 10 and 30 mg/mL ME1100 treatment groups compared with that in the placebo group. The number of bacteria in the lungs was significantly lower in the 30 mg/mL ME1100 treatment group at 6 h after the initial treatment, compared with all other groups. In the pharmacokinetic study, the Cmax in the 30 mg/mL ME1100 treatment group in the epithelial lining fluid (ELF) and plasma was 31.1 and 1.2 mg/L, respectively. Furthermore, we compared the efficacy of ME1100 with that of amikacin. Although there were no significant differences in ELF and plasma concentrations between 30 mg/mL of ME1100 and 30 mg/mL of amikacin, ME1100 significantly improved the survival rate compared with amikacin. Conclusions: The results of our study demonstrated the in vivo effectiveness of ME1100 and its superiority to amikacin

The definition of healthcare-associated pneumonia (HCAP) is insufficient for the medical environment in Japan: a comparison of HCAP and nursing and healthcare-associated pneumonia (NHCAP)

Healthcare-associated pneumonia (HCAP) is a new concept of pneumonia, which was proposed in the ATS/IDSA guidelines. The guidelines explain that HCAP patients should be treated with broad-spectrum antimicrobial drugs directed at multidrug-resistant pathogens. However, in Japan, there are many elderly people who received in-home care service. These patients seemed to be consistent with the concept of HCAP, but they did not meet the definition of HCAP. Therefore, the Japanese Respiratory Society modified the definition of HCAP according to the medical environmental in Japan. We retrospectively observed HCAP patients and nursing home and healthcare-associated pneumonia (NHCAP) patients who were hospitalized during 24 months at the Japanese Red Cross Nagasaki Genbaku Hospital (Nagasaki, Japan). Patient background, disease severity, identified pathogens, initial antibiotic regimens, and outcomes were compared. A total of 108 patients (77 HCAP and 31 NHCAP except HCAP patients) were evaluated. Of NHCAP except HCAP patients, 27 (87.1 %) were above 3 in the ECOG PS score. There were almost no significant differences between the two groups in characteristics, pneumonia severity, identified bacteria, initial antibiotic regimens, and response rate of initial antibiotic therapy. Although the in-hospital mortality of HCAP patients and NHCAP except HCAP patients was 9.1 % and 19.4 %, respectively, this difference did not reach statistical significance (P > 0.05). Our study suggested that, in the criteria of HCAP, some Japanese patients, who were consistent with the concept of HCAP, were classified as community-acquired pneumonia (CAP). Therefore, there is a need to change the definition of HCAP according to the medical environment in Japan

Had COVID-19 spread in the community before the first confirmed case in Nagasaki, Japan?

This retrospective study evaluated stored nasopharyngeal swab samples from Japanese patients with influenza-like illness during the 2019/2020 season. We aimed to determine whether COVID-19 had spread in the community before the first confirmed case. The period of influenza season during 2019/2020 in Nagasaki was shorter than in previous influenza seasons. When the first COVID-19 case was reported in Nagasaki prefecture, the number of influenza cases were very low. No positive results for SARS-CoV-2 were detected in 182 samples that were obtained from adult outpatients. Our results revealed no large-scale spread of COVID-19 in the community before the first confirmed case