Motoyasu Miyazaki

Presence of both heterogeneous vancomycin-intermediate resistance and β-lactam antibiotic-induced vancomycin resistance phenotypes is associated with the outcome in methicillin-resistant Staphylococcus aureus bloodstream infection

Abstract Background: Although the individual expression of heterogeneous vancomycin-intermediate resistance (hVISA) and β-lactam antibiotic-induced vancomycin resistance (BIVR) phenotypes has been associated with treatment failure and recurrence in methicillin-resistant Staphylococcus aureus (MRSA) infections, the effect of the co-expression of these phenotypic profiles on clinical outcome has not been fully elucidated. The aim of this study was to determine the impact of the combination of hVISA and BIVR phenotypes on the clinical outcome in MRSA bacteremia. Methods: One hundred and sixty-two MRSA blood isolates from a 21-y period, 1987–2007, were randomly selected. Screening for hVISA was done by the macromethod Etest and confirmed by population analysis profiles. BIVR was identified using Mu3 agar containing 4 μg/ml of vancomycin. Results: Thirty (18.5%) and 39 (24.1%) of the 162 MRSA blood isolates were positive for the hVISA and BIVR phenotypes, respectively. Eighteen (11.1%) isolates possessed both hVISA and BIVR phenotypes (hVISA(+)/BIVR(+)). In a subset of patients who received initial treatment with glycopeptides, only the patients whose isolates were hVISA(+)/BIVR(+) displayed a significantly higher mortality rate in comparison to those with non-hVISA(+)/BIVR(+) (80.0% vs 31.3%, p = 0.004). The presence of both hVISA and BIVR phenotypes was a predictor of mortality using a logistic regression analysis (p = 0.025). Conclusions: The combined phenotype of hVISA and BIVR was associated with a higher probability of mortality in patients with MRSA bacteremia. Further prospective studies are warranted to delineate the clinical significance of the combined phenotype of hVISA and BIVR.

Biofilm-Forming Methicillin-Resistant Staphylococcus aureus Survive in Kupffer Cells and Exhibit High Virulence in Mice

Although Staphylococcus aureus is part of the normal body flora, heavy usage of antibiotics has resulted in the emergence of methicillin-resistant strains (MRSA). MRSA can form biofilms and cause indwelling foreign body infections, bacteremia, soft tissue infections, endocarditis, and osteomyelitis. Using an in vitro assay, we screened 173 clinical blood isolates of MRSA and selected 20 high-biofilm formers (H-BF) and low-biofilm formers (L-BF). These were intravenously administered to mice and the general condition of mice, the distribution of bacteria, and biofilm in the liver, lung, spleen, and kidney were investigated. MRSA count was the highest in the liver, especially within Kupffer cells, which were positive for acid polysaccharides that are associated with intracellular biofilm. After 24 h, the general condition of the mice worsened significantly in the H-BF group. In the liver, bacterial deposition and aggregation and the biofilm-forming spot number were all significantly greater for H-BF group than for L-BF. CFU analysis revealed that bacteria in the H-BF group survived for long periods in the liver. These results indicate that the biofilm-forming ability of MRSA is a crucial factor for intracellular persistence, which could lead to chronic infections.

Rapid and easy detection of low-level resistance to vancomycin in methicillin-resistant Staphylococcus aureus by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Vancomycin-intermediately resistant Staphylococcus aureus (VISA) and heterogeneous VISA (hVISA) are associated with treatment failure. hVISA contains only a subpopulation of cells with increased minimal inhibitory concentrations, and its detection is problematic because it is classified as vancomycin-susceptible by standard susceptibility testing and the gold-standard method for its detection is impractical in clinical microbiology laboratories. Recently, a research group developed a machine-learning classifier to distinguish VISA and hVISA from vancomycin-susceptible S. aureus (VSSA) according to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) data. Nonetheless, the sensitivity of hVISA classification was found to be 76%, and the program was not completely automated with a graphical user interface. Here, we developed a more accurate machine-learning classifier for discrimination of hVISA from VSSA and VISA among MRSA isolates in Japanese hospitals by means of MALDI-TOF MS data. The classifier showed 99% sensitivity of hVISA classification. Furthermore, we clarified the procedures for preparing samples and obtaining MALDI-TOF MS data and developed all-in-one software, hVISA Classifier, with a graphical user interface that automates the classification and is easy for medical workers to use; it is publicly available at https://github.com/bioprojects/hVISAclassifier. This system is useful and practical for screening MRSA isolates for the hVISA phenotype in clinical microbiology laboratories and thus should improve treatment of MRSA infections.

Increased drug resistance of meticillin-resistant Staphylococcus aureus biofilms formed on a mouse dermal chip model

Purpose. Meticillin‐resistant Staphylococcus aureus (MRSA) biofilm formation in humans is of serious clinical concern. Previous in vitro studies have been performed with biofilms grown only on inorganic substrates; therefore, we investigated the vancomycin (VCM) resistance of MRSA biofilms grown on skin tissue. Methodology. We established a novel tissue substrate model, namely MRSA grown on segments of mouse skin tissue (dermal chips, DCs), and compared its resistance capacity against VCM with that of MRSA biofilms grown on plastic chips (PCs). Results/Key findings. For one MRSA isolate, we found that the VCM MIC was identical (1.56 &mgr;g ml−1) for planktonic cultures and for biofilms‐formed on PCs (PC‐BF), although the minimum bactericidal concentration (MBC) increased to 6.25 &mgr;g ml−1 in PC‐BF. On the contrary, the MIC and MBC for biofilms formed on DCs (DC‐BF) significantly increased (25 and 50 &mgr;g ml−1, respectively). Furthermore, the minimum biofilm‐eradicating concentration was higher for DC‐BF (100 &mgr;g ml−1) than for PC‐BF (25 &mgr;g ml−1). Using six MRSA strains, we found that in PC‐BF, the c.f.u. number decreased with increasing VCM concentration, whereas in DC‐BF, it greatly increased until the MIC was reached, accompanied by the formation of large colonies, thicker bacterial walls and the presence of many mitotic cells. Conclusion. Our results indicate that the VCM resistance of MRSA was greater in DC‐BF. We conclude that DCs may provide a specific environment for MRSA that enhances bacterial growth under cytotoxic VCM concentrations, and might be useful for the study of skin wound infections and the effects of antimicrobial drugs.

Association between medication adherence and illness perceptions in atrial fibrillation patients treated with direct oral anticoagulants: An observational cross-sectional pilot study

Objective The aim of this study was to examine the association between medication adherence and illness perceptions, and to explore the factors associated with poor medication adherence in atrial fibrillation (AF) patients receiving direct oral anticoagulants (DOACs) in a real-world clinical setting. Methods An observational cross-sectional pilot study was conducted at a single Japanese university hospital. One hundred and twenty-nine patients who were diagnosed with AF and who were taking DOACs were recruited from outpatients between January 4th and April 25th, 2017. We evaluated medication adherence to DOACs using the Morisky Medication Adherence Scale-8 (MMAS-8) and illness perceptions using the Brief Illness Perception Questionnaire (BIPQ). The patients’ characteristics and clinical data were collected from electronic medical records. Results Ninety-nine (76.7%) patients (male, n = 74; mean age, 71.4±9.8 years) participated in this study. According to the MMAS-8, 21 (21.2%) of the patients were classified into the poor adherence group (MMAS-8 score of <6), and 78 (78.8%) were classified into the good adherence group (MMAS-8 score of 6–8). A multivariate logistic regression analysis revealed that age (per year, odds ratio [OR] 0.912, 95% confidence interval [CI] 0.853–0.965, p = 0.001), a history of warfarin use (OR 0.181, 95% CI 0.033–0.764, p = 0.019), duration of DOAC exposure (per 100 days, OR 1.245, 95% CI 1.084–1.460, p = 0.001), and the BIPQ emotional response score (per 1 point, OR 1.235, 95% CI 1.015–1.527, p = 0.035) were significantly associated with poor medication adherence in AF patients receiving DOACs. Conclusion Poor medication adherence to DOACs was strongly associated with a stronger emotional response (i.e. stronger feelings of anger, anxiety, and depression), as well as younger age, the absence of a history of warfarin treatment, and longer DOAC exposure. Further evaluation of the factors associated with medication adherence in AF patients and the development and execution of strategies for improving poor adherence are warranted in the real-world clinical setting.

Adsorption of Nafamostat Mesilate on AN69ST Membranes: A Single‐Center Retrospective and in vitro Study

We examined whether AN69ST (acrylonitrile and methallyl sulfonate copolymer) membranes adsorb nafamostat mesilate. This study retrospectively analyzed 87 continuous hemodiafiltration sessions in vivo. We divided the continuous hemodiafiltration sessions into AN69ST and non‐AN69ST groups using the nafamostat mesilate dose and activated clotting time as indicators of nafamostat mesilate adsorption onto the membrane. Furthermore, we studied the in vitro adsorption of nafamostat mesilate from nafamostat mesilate solutions onto four different hemodialysis membranes. This in vivo study shows that nafamostat mesilate doses were significantly higher, but activated clotting times were shorter (P < 0.001) in the AN69ST group than in the non‐AN69ST group. These results suggest that AN69ST adsorbs nafamostat mesilate. Further, the in vitro experiments show that nafamostat mesilate adsorbs AN69ST on membranes significantly more than the other membranes tested. These in vitro and clinical findings provide evidence that AN69ST may adsorb nafamostat mesilate.

Prevalence of Slow-Growth Vancomycin Nonsusceptibility in Methicillin-Resistant Staphylococcus aureus

ABSTRACT We previously reported a novel phenotype of vancomycin-intermediate Staphylococcus aureus (VISA), i.e., “slow VISA,” whose colonies appear only after 72 h of incubation. Slow-VISA strains can be difficult to detect because prolonged incubation is required and the phenotype is unstable. To develop a method for detection of slow-VISA isolates, we studied 23 slow-VISA isolates derived from the heterogeneous VISA (hVISA) clinical strain Mu3. We identified single nucleotide polymorphisms (SNPs) in genes involved in various pathways which have been implicated in the stringent response, such as purine/pyrimidine synthesis, cell metabolism, and cell wall peptidoglycan synthesis. We found that mupirocin, which also induces the stringent response, caused stable expression of vancomycin resistance. On the basis of these results, we developed a method for detection of slow-VISA strains by use of 0.032 μg/ml mupirocin (Yuki Katayama, 7 March 2017, patent application PCT/JP2017/008975). Using this method, we detected 53 (15.6%) slow-VISA isolates among clinical methicillin-resistant S. aureus (MRSA) isolates. In contrast, the VISA phenotype was detected in fewer than 1% of isolates. Deep-sequencing analysis showed that slow-VISA clones are present in small numbers among hVISA isolates and proliferate in the presence of vancomycin. This slow-VISA subpopulation may account in part for the recurrence and persistence of MRSA infection.