Immunomics analysis of rheumatoid arthritis identified precursor dendritic cells as a key cell subset of treatment resistance.

OBJECTIVES: Little is known about the immunology underlying variable treatment response in rheumatoid arthritis (RA). We performed large-scale transcriptome analyses of peripheral blood immune cell subsets to identify immune cells that predict treatment resistance. METHODS: We isolated 18 peripheral blood immune cell subsets of 55 patients with RA requiring addition of new treatment and 39 healthy controls, and performed RNA sequencing. Transcriptome changes in RA and treatment effects were systematically characterised. Association between immune cell gene modules and treatment resistance was evaluated. We validated predictive value of identified parameters for treatment resistance using quantitative PCR (qPCR) and mass cytometric analysis cohorts. We also characterised the identified population by synovial single cell RNA-sequencing analysis. RESULTS: Immune cells of patients with RA were characterised by enhanced interferon and IL6-JAK-STAT3 signalling that demonstrate partial normalisation after treatment. A gene expression module of plasmacytoid dendritic cells (pDC) reflecting the expansion of dendritic cell precursors (pre-DC) exhibited strongest association with treatment resistance. Type I interferon signalling was negatively correlated to pre-DC gene expression. qPCR and mass cytometric analysis in independent cohorts validated that the pre-DC associated gene expression and the proportion of pre-DC were significantly higher before treatment in treatment-resistant patients. A cluster of synovial DCs showed both features of pre-DC and pro-inflammatory conventional DC2s. CONCLUSIONS: An increase in pre-DC in peripheral blood predicted RA treatment resistance. Pre-DC could have pathophysiological relevance to RA treatment response.

Utility of nailfold capillary assessment for predicting psoriatic arthritis based on a prospective observational cohort study.

OBJECTIVES: PsA is one of the most serious comorbidities associated with psoriasis. While the early intervention in PsA is demanded, risk factors of PsA development are not well-known. This is the first prospective study to evaluate the clinical significance of nailfold capillary (NFC) changes in patients with psoriasis. METHODS: We conducted a prospective cohort study in a population of 449 psoriasis patients who had not been treated with systemic therapy or topical finger therapy. NFCs were observed by dermoscopy and capillaroscopy, and the correlation of NFC abnormalities, including nailfold bleeding (NFB) and enlarged capillaries, with the prevalence of PsA, incidence of new PsA, and serum levels of TNF-a, IL-17A and IL-23 were analysed. RESULTS: Detailed examination at the time of inclusion revealed that of 449 patients, 236 had Psoriasis vulgaris (PsV) and 213 had PsA. Both NFB and enlarged capillaries were significantly more frequent in patients with PsA (34.7% vs 84.5%, P < 0.0001; 25.4% vs 100%, P < 0.0001). In addition, PsV patients were prospectively observed before they developed PsA (mean 21 months, 95% CI 2, 77 months). Multivariate analysis suggested that the appearance of NFB and enlarged capillaries was a predictor of PsA development (HR 2.75, 95% CI 1.38, 5.47 and HR 4.49, 95% CI 2.25, 8.96, respectively). The degree of NFC abnormalities also correlated with the severity of PsA and serum cytokine levels. CONCLUSIONS: NFC abnormalities were suggested to be a predictor of PsA in psoriasis patients, and at the same time, its degree could be an indicator of disease severity.

Utility of nailfold capillary assessment for predicting pustulotic arthro-osteitis in palmoplantar pustulosis based on a prospective cohort study.

BACKGROUND: Pustulotic arthro-osteitis (PAO) is 1 of the most serious comorbidities associated with palmoplantar pustulosis (PPP). Risk factors of PAO development are not well-known. OBJECTIVE: To evaluate the clinical significance of nailfold capillary (NFC) changes in patients with PPP. METHODS: We conducted a prospective cohort study in a population of 102 PPP patients. Correlations of NFC abnormalities, including nailfold bleeding and enlarged capillaries, with the prevalence of PAO, the incidence of new PAO, and serum levels of cytokines were analyzed. RESULTS: Detailed examination revealed that of 102 PPP patients, 52 without PAO and 50 with PAO. Both nailfold bleeding and enlarged capillaries were significantly more frequent in patients with PAO (50.0% vs 92.0%, P < .0001; 50.0% vs 94.0%, P < .0001). In addition, PPP patients without PAO were prospectively observed before they developed PAO (mean 28 months [1-52 months]). Multivariate analysis suggested that these NFC abnormalities were predictors of PAO development (hazard ratio 3.37, 95% confidence interval 1.13-10.07; 3.37, 1.13-10.07) and guselkumab prevent PAO development (0.093, 0.012-0.76). The degree of NFC abnormalities correlated with the severity of PAO and serum cytokine levels. LIMITATIONS: All participants were Japanese. CONCLUSION: NFC abnormalities could be predictors of PAO in PPP patients, and their degree indicators of disease severity.

Decreased peripheral blood memory B cells are associated with the presence of interstitial lung disease in rheumatoid arthritis: a case-control study.

OBJECTIVES: Interstitial lung disease sometimes occurs in rheumatoid arthritis patients. Although the underlying immunological mechanisms responsible for interstitial lung disease associated with rheumatoid arthritis have not yet been clarified, some reports have suggested possible roles of B cells. To examine the role of B-cell subsets in interstitial lung disease in rheumatoid arthritis patients, we analyzed peripheral blood B-cell subsets. METHODS: We analyzed the frequencies of the peripheral blood B-cell subsets by flow cytometry in rheumatoid arthritis patients with and without interstitial lung disease (n = 16 and 81, respectively) and in healthy donors (n = 110) by high-resolution computed tomography. RESULTS: Compared with healthy donors, rheumatoid arthritis patients showed statistically higher frequencies of naive B cells and lower frequencies of memory B cells. Moreover, the frequencies of memory B cells were lower in rheumatoid arthritis patients with interstitial lung disease than in those without. Multivariate analysis showed that the frequency of memory B cells, particularly switched memory B cells, was significantly decreased in rheumatoid arthritis patients with interstitial lung disease, even after adjusting for prednisolone dose. CONCLUSIONS: We suspect memory B cells play important roles in interstitial lung disease associated with rheumatoid arthritis.

Factors associated with discontinuation of glucocorticoids after starting biological disease-modifying antirheumatic drugs in rheumatoid arthritis patients.

Objectives: Glucocorticoids (GCs) are effective treatments for rheumatoid arthritis (RA) but long-term use has adverse effects. This study aimed to elucidate whether GCs can be discontinued by introducing biological disease-modifying antirheumatic drugs (bDMARDs) and the factors influencing the outcome.Method: We included RA patients who had been orally taking GCs at the initiation of bDMARDs. The changes in GC dose after starting bDMARDs were evaluated and the GC discontinuation rate was analyzed using Kaplan-Meier analysis. The factors associated with discontinuation of GCs were assessed by Cox hazard models.Results: Eighty RA patients were included in the study. The dosage of oral prednisolone (PSL) was significantly reduced from 5.0 to 3.0 mg/day by 3 months (p = .013). GCs were discontinued in 31.3% of patients and the median time until GC discontinuation was 10.1 months. The GC discontinuation rate was significantly higher in patients with Class 1 and 2 (p = .024), with an initial PSL dose <5 mg/day (p = .040), and with low DAS28(ESR) (p = .038). In multivariate analyses, higher DAS28(ESR) (odds ratio, 0.200; p = .039), and higher PSL dose (odds ratio, 0.748; p = .029) were significantly associated with less frequent GC discontinuation.Conclusion: DAS28(ESR) high and PSL dose were factors associated with discontinuation of GC use after starting bDMARDs.

Sinus bradycardia after intravenous pulse methylprednisolone therapy in patients with systemic lupus erythematosus.

Sinus bradycardia is reported as an adverse effect of high-dose glucocorticoid therapy. We report three cases of systemic lupus erythematosus, wherein intravenous pulse methylprednisolone was administered. The patients' average baseline heart rate was 72 beats/min, which decreased 30% from baseline at 61 h after beginning the therapy. The average minimum heart rate was 38 beats/min, and this rate continued for 169 h on average. No other causes for bradycardia were found, suggesting that the administration of glucocorticoid pulse therapy resulted in decreased heart rate.

A gene module associated with dysregulated TCR signaling pathways in CD4+ T cell subsets in rheumatoid arthritis.

We analyzed the transcriptome of detailed CD4+ T cell subsets including them after abatacept treatment, and examined the difference among CD4+ T cell subsets and identified gene sets that are closely associated disease activity and abatacept treatment. Seven CD4+ T cell subsets (naive, Th1, Th17, Th1/17, nonTh1/17, Tfh and Treg) were sorted from PBMCs taken from 10 RA patients and 10 healthy controls, and three RA patients donated samples before and 6 months after abatacept treatment. Paired-end RNA sequencing was performed using HiSeq 2500. A total of 149 samples except for 12 outliers were analyzed. Overview of expression pattern of RA revealed that administration of abatacept exerts a large shift toward the expression pattern of HC. Most of differentially expressed gene (DEG) upregulated in RA (n = 1776) were downregulated with abatacept treatment (n = 1349). Inversely, most of DEG downregulated in RA (n = 1860) were upregulated with abatacept treatment (n = 1294). This DEG-based analysis revealed shared pathway changes in RA CD4+ T cell subsets. Knowledge-based pathway analysis revealed the upregulation of activation-related pathways in RA that was substantially ameliorated by abatacept. Weighted gene co-expression network analysis (WGCNA) evaluated CD4+ T cells collectively and identified a gene module that consisted of 227 genes and was correlated with DAS28-CRP (Spearman's rho = 0.46, p = 4 × 10-9) and abatacept administration (Spearman's rho = -0.91, p = 5 × 10-57). The most highly connected 30 genes of this module included ZAP70 and JAK3, and pathway analysis of this module revealed dysregulation of the TCR signaling pathway network, which was ameliorated by abatacept.

Polymorphic lymphoproliferative disorders in patients with rheumatoid arthritis are associated with a better clinical outcome.

OBJECTIVES: The characteristics of lymphoproliferative disorders (LPD) in patients with rheumatoid arthritis (RA) remain unclear. Therefore, we retrospectively analyzed the clinical characteristics of these patients in our department. METHODS: Twenty RA patients who developed LPD between April 2003 and August 2016 in our department were analyzed. RESULTS: All of the RA patients who developed LPD had been treated with methotrexate (MTX). The median weekly and total dosages of MTX were 6.8 mg/week and 2530 mg, respectively. The median duration of MTX administration was eight years. Nineteen patients (95%) achieved complete remission (CR) and 15 (75%) achieved CR with MTX cessation alone. Based on the pathological findings, we divided MTX-associated LPD patients into two groups (n = 16); polymorphic LPD (31%) and other groups. CR with MTX cessation alone was achieved in 5 (100%) and 6 (54.5%) patients in the polymorphic LPD and other groups, respectively (p = .12). Moreover, the duration from the cessation of MTX to CR was significantly shorter in the polymorphic LPD group than in the other group (5.3 months vs 12.6 months, p = .01, respectively). CONCLUSION: Polymorphic LPD, which was the most frequent pathological diagnosis in this cohort, was associated with a higher incidence of CR and a significantly shorter duration to CR.

Enhanced gut homing receptor expression of unswitched memory B cells in rheumatoid arthritis.

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Digestion of chrysanthemum stunt viroid by leaf extracts of Capsicum chinense indicates strong RNA-digesting activity.

KEY MESSAGE: CSVd could not infect Nicotiana benthamiana when the plants were pretreated with crude leaf extract of Capsicum chinense 'Sy-2'. C. chinense leaves were revealed to contain strong RNA-digesting activity. Several studies have identified active antiviral and antiviroid agents in plants. Capsicum plants are known to contain antiviral agents, but the mechanism of their activity has not been determined. We aimed to elucidate the mechanism of Capsicum extract's antiviroid activity. Chrysanthemum stunt viroid (CSVd) was inoculated into Nicotiana benthamiana plants before or after treating the plants with a leaf extract of Capsicum chinense 'Sy-2'. CSVd infection was determined using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) 3 weeks after inoculation. When Capsicum extract was sprayed or painted onto N. benthamiana before inoculation, it was effective in preventing infection by CSVd. To evaluate CSVd digestion activity in leaf extracts, CSVd was mixed with leaf extracts of Mirabilis, Phytolacca, Pelargonium and Capsicum. CSVd-digesting activities were examined by quantifying undigested CSVd using qRT-PCR, and RNA gel blotting permitted visualization of the digested CSVd. Only Capsicum leaf extract digested CSVd, and in the Capsicum treatment, small digested CSVd products were detected by RNA gel blot analysis. When the digesting experiment was performed for various cultivars and species of Capsicum, only cultivars of C. chinense showed strong CSVd-digesting activity. Our observations indicated that Capsicum extract contains strong RNA-digesting activity, leading to the conclusion that this activity is the main mechanism for protection from infection by CSVd through spraying or painting before inoculation. To our knowledge, this is the first report of a strong RNA-digesting activity by a plant extract.

An elderly woman with peripheral spondyloarthritis with aortitis.

A 75-year-old woman was admitted to our department because of backache and multiple joint pain. Serum C-reactive protein (CRP) level was 6.8 mg/dL, and serum rheumatoid factor and anti-citrullinated peptide antibody were negative. Magnetic resonance imaging (MRI) showed bone edema and synovitis of the sacroiliac joints. (18)Fluorodeoxyglucose-positron emission tomography ((18)FDG-PET) showed increased uptakes in the bilateral elbow, shoulder, sternoclavicular, hip, and sacroiliac joints. In addition, increased uptakes were also observed in the bilateral cervical and subclavian arteries, and the thoracic aorta. Moreover, inflammation of the vascular walls and an aneurism in the right subclavian artery were observed on MRI. The patient's HLA type was HLA-B48 and B60. According to the Assessment of Spondyloarthritis (ASAS) classification criteria, peripheral spondyloarthropathy (SpA) was also diagnosed. Although the diagnostic criteria of Takayasu aortitis or giant cell aortitis were not fulfilled, we thought that active aortitis was also involved, and high-dose prednisolone was started. The patient's symptoms were diminished immediately, and CRP levels returned to normal. Although the etiology of the aortitis was not certain, this is the first report of late-onset peripheral SpA with aortitis, diagnosed by (18)FDG-PET and MRI. We recommended that it is important to evaluate the aortic involvement in late-onset SpA patients, when elevated systemic inflammation is observed.

Psoriasis treatment and biologic switching: The association with clinical characteristics and laboratory biomarkers over a 13-year retrospective study.

The advent of biologics has greatly improved patient outcomes, yet some patients are compelled to switch therapies. Predicting these therapeutic failures is important; however, the factors associated with switching biologics have not been fully explored. This study examined patterns and determinants of biologics switching in psoriasis treatment retrospectively over 13 years. We focused on the association between clinical characteristics, basal laboratory data, and frequency of biologics switching. The findings revealed that elevated Psoriasis Area Severity Index scores and the presence of arthritis were observed in patients who experienced two or more treatment switches compared with those without treatment switches. Moreover, neutrophil to lymphocyte ratio was associated with higher biologics switching rates, indicating that systemic inflammation significantly impacts treatment adherence. A treatment approach, taking into account both the clinical presentation and inflammatory biomarkers, may be important for optimizing patient management in psoriasis.

Prevalence of antiphospholipid autoantibodies associated with biologics treatment for psoriasis.

Psoriasis is a chronic inflammatory disease that sometimes necessitates therapeutic intervention with biologics. Autoantibody production during treatment with tumor necrosis factor (TNF) inhibitors is a recognized phenomenon, however, the production of autoantibodies associated with antiphospholipid syndrome (APS) has not been comprehensively evaluated in patients with psoriasis. This study was conducted to assess the prevalence of APS-associated autoantibodies in patients with psoriasis treated with different biologics and to investigate the potential associations between autoantibody production and clinical or serological parameters. Patients with psoriasis undergoing biologics treatments were enrolled in this study, and were categorized based on the type of biologics administered, TNF, interleukin (IL)-17, or IL-23 inhibitors. Clinical and serological data were collected and analyzed in conjunction with data on APS autoantibodies. TNF inhibitors were associated with a higher frequency of APS autoantibodies compared to IL-17 and IL-23 inhibitors. Notably, the presence of APS autoantibodies correlated with concurrent arthritis and higher disease severity at treatment initiation in patients treated with TNF inhibitors. Elevated Psoriasis Area and Severity Index scores and anti-nuclear antibody titers higher than × 320 were predictors of APS autoantibody production. Despite the higher autoantibody rates, clinical symptoms of APS were absent in these patients. This study provides the first comprehensive evidence of an increased frequency of APS autoantibodies associated with TNF inhibitor treatment in patients with psoriasis. The observed association between APS autoantibody positivity and TNF inhibitor treatment or clinical parameters suggests a potential immunomodulatory interplay between autoimmunity and inflammation in the pathogenesis of psoriasis.

Long-term therapeutic effects and safety of tacrolimus added to methotrexate in patients with rheumatoid arthritis.

To assess the long-term safety and efficacy of tacrolimus (TAC) used in combination with oral methotrexate (MTX) in patients with rheumatoid arthritis (RA) whose disease remains active despite treatment with MTX alone. The clinical courses of 24 RA patients who received TAC added to MTX from a single center were analyzed retrospectively. The disease activity was evaluated by the DAS28-ESR(3) every 12 months after the addition of TAC, and side effects were evaluated for 3 years. At 3 years after starting the treatment, TAC was still being used by 19 patients (79 %). The causes of discontinuation were an inadequate response (3 cases), oral ulcers and elevation of creatinine (1 case), and worsening of interstitial pneumonia (1 case). No death was registered. The DAS28-ESR(3) was decreased from 4.81 to 3.41 after 3 years of treatment. The doses of prednisolone were decreased from 5.1 mg/day to 3.2 mg/day after 3 years. In patients whose active RA persists despite treatment with MTX, TAC in combination with MTX is safe and well tolerated and provided clinical benefit for a long time in this single-center retrospective study. Further studies are required to confirm the safety and efficacy of this combination therapy.

Rapid bactericidal activity of sitafloxacin against Streptococcus pneumoniae.

The initial bactericidal activity of quinolones against Streptococcus pneumoniae at the concentration equivalent to their respective peak serum concentration (C(max)) and free drug fraction of C(max) (fC(max)) were investigated. The bactericidal activity of sitafloxacin (STFX), levofloxacin (LVFX), moxifloxacin (MFLX), and garenoxacin (GRNX) were compared by determining the actual killing of bacteria at C(max) and fC(max) for 1 and 2 hours based on the Japanese maximum dose per administration (100, 500, 400, and 400 mg, respectively). Against 4 quinolone-susceptible clinical isolates (wild-type), STFX with C(max) and fC(max) exhibited the most rapid bactericidal activity resulting in an average reduction of > or = 3.0 log10 colony forming units (CFU)/ mL in 1 hour. STFX with C(max) and fC(max) also showed the most rapid and potent bactericidal activity against 9 clinical isolates with single par (C/E) mutation, resulting in > or = 3.0 log10 CFU/mL average reduction in viable cells in 1 hour. STFX showed a statistically significant advantage in initial bactericidal activity over other quinolones for single mutants (P < 0.001). The propensity that the difference in the initial bactericidal activity between STFX and other quinolones was higher in single mutants than wild-type strains, was confirmed using S. pneumoniae ATCC49619 (wild-type) and its laboratory single parC mutant. As a result, STFX showed a similar rapid and potent initial bactericidal activity against both strains, while initial bactericidal activity for other quinolones was significantly reduced in the single mutant (P < 0.05). In conclusion, STFX has the most rapid and potent initial bactericidal activity against wild-type and single mutants of S. pneumoniae and its bactericidal activity is not affected by the presence of a single par mutation compared to LVFX, MFLX, and GRNX.

Successful treatment with tocilizumab in a case of Cogan's syndrome complicated with aortitis.

A 69-year-old man with sensorineural hearing loss and iritis was diagnosed with atypical Cogan's syndrome. He had several systematic manifestations: aortitis, meningitis, panniculitis and seronegative arthritis. Remission induced by treatment with high doses of prednisolone was followed by relapse within 1 year. Although his condition was resistant to various immunosuppressive drugs, including methotrexate, cyclosporine, azathioprine and adalimumab, his symptoms, inflammatory response and quality of life measures were successfully improved by tocilizumab, a humanized anti-interleukin-6 receptor antibody.

Carriers of HLA-DRB1*04:05 have a better clinical response to abatacept in rheumatoid arthritis.

HLA-DRB1 shared epitope risk alleles are the strongest genetic risk factors for rheumatoid arthritis (RA) and potential biomarkers for treatment response to biological disease-modifying antirheumatic drugs (bDMARDs). This study aimed to investigate the association between treatment response and individual HLA-DRB1 alleles in RA patients receiving different bDMARDs. We recruited 106 patients with active RA who had started abatacept, tocilizumab, or TNF inhibitors as a first-line bDMARDs. We examined the relationship between Simplified Disease Activity Index (SDAI) improvement at 3 months and HLA-DRB1 allele carriage. The results revealed that the HLA-DRB1*04:05 allele, a shared-epitope allele, was significantly associated with better SDAI improvement only after abatacept treatment (SDAI improvement 28.5% without the allele vs 59.8% with allele, p = 0.003). However, no significant association was found with other treatments. Both multivariate linear regression and mediation analysis confirmed that the HLA-DRB1*04:05 allele was independently associated with abatacept treatment response, regardless of anti-CCP antibody titers. The study concluded that in patients with RA receiving their first-line bDMARD treatment, carrying the HLA-DRB1*04:05 allele was associated with better SDAI improvement specifically in abatacept-treated patients. These disease-risk HLA alleles have the potential to serve as genomic biomarkers for predicting treatment response with co-stimulation blockage therapy.

The optimal use of tildrakizumab in the elderly via improvement of Treg function and its preventive effect of psoriatic arthritis.

Introduction: As a form of precision medicine, this study aimed to investigate the specific patient population that would derive the greatest benefit from tildrakizumab, as well as the mechanism of action and efficacy of tildrakizumab in reducing the occurrence of psoriatic arthritis (PsA). Methods: To achieve this, a multi-center, prospective cohort study was conducted, involving a population of 246 psoriasis patients who had not received any systemic therapy or topical finger therapy between January 2020 and April 2023. Two independent clinicians, who were blinded to the study, analyzed nailfold capillary (NFC) abnormalities, such as nailfold bleeding (NFB) and enlarged capillaries, as well as the incidence of new PsA. Additionally, the factors that determined the response of psoriasis after seven months of tildrakizumab treatment were examined. The study also examined the quantity and role of regulatory T cells (Tregs) and T helper 17 cells both pre- and post-treatment. Results: The severity of psoriasis, as measured by the Psoriasis Area and Severity Index (PASI), was found to be more pronounced in the tildrakizumab group (n=20) in comparison to the topical group (n=226). At 7 months after tildrakizumab treatment, multivariate analysis showed that those 65 years and older had a significantly better response to treatment in those achieved PASI clear or PASI 2 or less (Likelihood ratio (LR) 16.15, p<0.0001; LR 6. 16, p=0.01). Tildrakizumab improved the number and function of Tregs, which had been reduced by aging. Tildrakizumab demonstrated significant efficacy in improving various pathological factors associated with PsA. These factors include the reduction of NFB, enlargement of capillaries, and inhibition of PsA progression. The hazard ratio for progression to PsA was found to be 0.06 (95% confidence interval: 0.0007-0.46, p=0.007), indicating a substantial reduction in the risk of developing PsA. Discussion: Tildrakizumab's effectiveness in improving skin lesions can be attributed to its ability to enhance the number and function of Tregs, which are known to decline with age. Furthermore, the drug's positive impact on NFB activity and capillary enlargement, both of which are recognized as risk factors for PsA, further contribute to its inhibitory effect on PsA progression.

[Bactericidal effect of levofloxacin injection in combination with meropenem against Pseudomonas aeruginosa using an in vitro simulation model with a hollow fiber system].

An in vitro human plasma concentration simulation model with a hollow fiber system was established and used to evaluate the bactericidal effect of levofloxacin (LVFX) 500mg q.d. in combination with meropenem (MEPM) 1000mg t.i.d. against Pseudomonas aeruginosa. Six clinical isolates of P. aeruginosa which had MEPM MICs of 2 - 16 microg/mL, LVFX MICs of 2 microg/mL, and fractional inhibitory concentration (FIC) indices by the in vitro checkerboard method of 0.625-1 were used. In the treatment with MEPM alone, initial viable counts (10(6)-10(7) CFU/ mL) decreased, but did not reach below the detection limit (100 CFU/mL) and the regrowth of bacteria was observed. In the treatment with LVFX alone, viable counts decreased once below the detection limit, although increased after treatment for 24 hours. On the other hand, in the treatment with LVFX-MEPM combination, more potent bactericidal effects were observed compared to LVFX or MEPM alone in all strains. Especially, in the strains with MEPM MICs of 2 and 4 microg/mL, viable counts rapidly decreased below the detection limit and no regrowth was observed until 24 hours. These results suggested that LVFX-MEPM has a potential to be an effective combination against P. aeruginosa by synergistic rapid bactericidal action in clinical settings, even in the strain against which no significant synergy is confirmed by the traditional in vitro checkerboard method.