Clinical characteristics and analysis of prognostic factors in methicillin-resistant Staphylococcus aureus endocarditis: A retrospective multicenter study in Japan.

BACKGROUND: Infective endocarditis (IE) caused by MRSA (methicillin-resistant Staphylococcus aureus) is associated with a high mortality rate. This study aimed to elucidate the characteristics of patients with MRSA-IE in Japan and identify the factors associated with prognosis. METHODS: This retrospective study included patients with a confirmed diagnosis of IE caused by MRSA, between January 2015 and April 2019. RESULTS: A total of 65 patients from 19 centers were included, with a mean age of 67 years and 26 % were female. Fifty percent of the patients with IE were had nosocomial infections and 25 % had prosthetic valve involvement. The most common comorbidities were hemodialysis (20 %) and diabetes (20 %). Congestive heart failure was present in 86 % of patients (NYHA class I, II: 48 %; III, IV: 38 %). The 30-day and in-hospital mortality rates were 29 % and 46 %, respectively. Multi-organ failure was the primary cause of death, accounting for 43 % of all causes of death. Prognostic factors for in-hospital mortality were age, disseminated intravascular coagulation, daptomycin and/or linezolid as initial antibiotic therapy, and surgery. Surgical treatment was associated with a lower mortality rate (odds ratio [OR], 0.026; 95 % confidence interval [CI], 0.002-0.382; p = 0.008 for 30-day mortality and OR, 0.130; 95 % CI; 0.029-0.584; p = 0.008 for in-hospital mortality). CONCLUSION: Mortality due to MRSA-IE remains high. Surgical treatment is a significant prognostic predictor of MRSA-IE.

Direct Species Identification in Positive Blood Culture Bottles From Patients With Hematologic Malignancies.

Background In patients with hematologic malignancies, faster species identification is particularly important in the management of bloodstream infection because of their immunocompromised and neutropenic status. In the present study, we analyzed direct species identification in patients with hematologic malignancies, and the factors that might influence the results of species identification. Methods We performed direct species identification using a Sepsityper® kit (Bruker Corporation, Billerica, Massachusetts, United States) and compared the results with a conventional method in patients with hematologic malignancies. Forty-five positive blood culture bottles containing single microorganisms from 37 patients were analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). And patients' clinical data were compared between the groups with spectral scores at acceptable and unacceptable levels. Results Direct species identification correctly identified 42 of 45 isolates and three were misidentified. While 35 of 45 isolates showed a spectral score ≥1.7 (acceptable identification), 10 isolates had a spectral score <1.7 (unacceptable identification) including three misidentified isolates. The group with a spectral score ≥1.7 had significantly lower white blood cell (p<0.01), neutrophil (p<0.01), and platelet (p<0.01) counts in addition to more frequent central venous (CV) line insertion (p=0.01). Multivariate analysis revealed that pathogen type (gram-positive or negative) and CV line insertion were associated with spectral scores. Conclusion Direct species identification using the Sepsityper kit is an upcoming approach for blood culture bottles, which were flagged as positive even in patients with hematologic malignancies when the spectral score was ≥ 1.7. Our study also indicates that direct identification is more accurate in patients with CV lines, and may be less accurate when gram-positive bacteria are detected.

Antimicrobial therapy and outcome of methicillin-resistant Staphylococcus aureus endocarditis: A retrospective multicenter study in Japan.

BACKGROUND: MRSA (methicillin-resistant Staphylococcus aureus)-infective endocarditis (IE) is associated with high morbidity and mortality. This study aimed to assess data from patients with MRSA-IE across multiple facilities in Japan, with a specific focus on antimicrobial therapy and prognosis. METHODS: This retrospective study enrolled patients with a confirmed diagnosis of IE attributed to MRSA, spanning the period from January 2015 to April 2019. RESULTS: Sixty-four patients from 19 centers were included, with a median age of 67 years. The overall mortality rate was 28.1% at 30 days, with an in-hospital mortality of 45.3%. The most frequently chosen initial anti-MRSA agents were glycopeptide in 67.2% of cases. Daptomycin and linezolid were selected as initial therapy in 23.4% and 17.2% of cases, respectively. Approximately 40% of all patients underwent medication changes due to difficulty in controlling infection or drug-related side effects. Significant prognostic factors by multivariable analysis were DIC for 30-day mortality and surgical treatment for 30-day and in-hospital mortality. For vancomycin as initial monotherapy, there was a trend toward a worse prognosis for 30-day and in-hospital mortality (OR, 6.29; 95%CI, 1.00-39.65; p = 0.050, OR, 3.61; 95%CI, 0.93-14.00; p = 0.064). Regarding the choice of initial antibiotic therapy, statistical analysis did not show significant differences in prognosis. CONCLUSION: Glycopeptide and daptomycin were the preferred antibiotics for the initial therapy of MRSA-IE. Antimicrobial regimens were changed for various reasons. Prognosis was not significantly affected by choice of antibiotic therapy (glycopeptide, daptomycin, linezolid), but further studies are needed to determine which antimicrobials are optimal as first-line agents.

COVID-19 and Listeria Meningitis Treated by Ampicillin, Sulfamethoxazole/Trimethoprim and Meropenem.

Background: Secondary bacterial infection was initially rare in SARS-CoV-2 infectious disease (COVID-19) patients, but COVID-19-associated bacterial infectious diseases have recently been increasing. Furthermore, it might be difficult to distinguish COVID-19 from bacterial meningitis by the symptoms, and one might be uncertain about antibiotic therapy for Listeria meningitis infection-typically caused by eating contaminated food-in elderly persons and pregnant women. Case Report: A 96-year-old woman who had been living alone was found to have SARS-CoV-2 infection in February 2023. She was admitted to our hospital with high fever and disturbance of consciousness and was started on treatment with remdesivir. Two days later, her consciousness was still disturbed, and she was found to have a stiff neck. In addition, increased white blood cell counts and C-reactive protein suggested bacterial infection. Therefore, a lumbar puncture was done, and Listeria monocytogenes was ultimately isolated from blood cultures and its genetic material was detected in cerebrospinal fluid. She had previously eaten refrigerated food and cheese products. Intravenous ampicillin 1.0 g 6×/day was started, but one week later, loss of consciousness continued, and the cerebrospinal findings were not improved, although nasal swab became negative for SARS-CoV-2. Intravenous sulfamethoxazole/trimethoprim (ST) 80/400 mg 3×/day was added, and her consciousness and fever improved by one week later. A drug rash appeared after ST was started, and she was switched to meropenem. Her condition finally improved. Conclusion: COVID-19-associated secondary listeria infection was found in an elderly woman. She was treated with not only ampicillin, but also ST and meropenem. Meningitis caused by Listeria monocytogenes should be considered as a secondary complication and carefully treated with antibiotics during the period of the COVID-19 pandemic.

Effectiveness and Safety of Linezolid Versus Vancomycin, Teicoplanin, or Daptomycin against Methicillin-Resistant Staphylococcus aureus Bacteremia: A Systematic Review and Meta-Analysis.

Vancomycin (VCM) and daptomycin (DAP) are standard therapies for methicillin-resistant Staphylococcus aureus (MRSA) bacteremia, despite concerns regarding clinical utility and growing resistance. Linezolid (LZD) affords superior tissue penetration to VCM or DAP and has been successfully used as salvage therapy for persistent MRSA bacteremia, indicating its utility as a first-choice drug against MRSA bacteremia. In a systematic review and meta-analysis, we compared the effectiveness and safety of LZD with VCM, teicoplanin (TEIC), or DAP in patients with MRSA bacteremia. We evaluated all-cause mortality as the primary effectiveness outcome, clinical and microbiological cure, hospital length of stay, recurrence, and 90-day readmission rates as secondary effectiveness outcomes, and drug-related adverse effects as primary safety outcomes. We identified 5328 patients across 2 randomized controlled trials (RCTs), 1 pooled analysis of 5 RCTs, 1 subgroup analysis (1 RCT), and 5 case-control and cohort studies (CSs). Primary and secondary effectiveness outcomes were comparable between patients treated with LZD versus VCM, TEIC, or DAP in RCT-based studies and CSs. There was no difference in adverse event incidence between LZD and comparators. These findings suggest that LZD could be a potential first-line drug against MRSA bacteremia as well as VCM or DAP.

Comparison of the efficacy and safety of standard- and high-dose daptomycin: A systematic review and meta-analysis.

AIMS: Standard doses of daptomycin at 4 and 6 mg/kg were used for the treatment of skin and soft tissue for infections and bacteraemia, respectively. However, increased doses of daptomycin are recommended for complicated infections by Gram-positive organisms. METHODS: A systematic review was conducted using 4 databases. We compared treatment success between standard-dose (SD, 4-6 mg/kg) and high-dose (HD, >6 mg/kg) daptomycin in patients with all-cause bacteraemia, complicated bacteraemia, infective endocarditis, osteomyelitis and foreign body/prosthetic infection as the primary outcome. We also compared the success between SD and HD2 (≥8 mg/kg) daptomycin treatments in patients with these diseases as the secondary outcome. The incidence of creatine phosphokinase (CPK) elevation was evaluated as safety. RESULTS: In patients with complicated bacteraemia and infective endocarditis, the treatment success was significantly lower in the SD group than in the HD group (odds ratio [OR] 0.48, 95% confidence interval [CI] 0.30-0.76 and OR 0.50, 95% CI 0.30-0.82) and HD2 group (OR 0.38, 95% CI 0.21-0.69 and OR 0.30, 95% CI 0.15-0.60), respectively. A significant difference was demonstrated only in the HD2 group in patients with bacteraemia, including simple infection. SD did not decrease the success rate for the treatment of osteomyelitis and foreign body/prosthetic infection. The incidence of elevated CPK was significantly lower in SD group than in HD group. CONCLUSION: SD daptomycin was associated with significantly lower treatment success than HD in patients with complicated bacteraemia/infective endocarditis. The CPK elevation should be considered in patients treated with high daptomycin doses.

Otitis externa caused by Malassezia slooffiae complicated with mastoiditis: A case report.

Herein, we report a case of otitis externa caused by Malassezia slooffiae complicated with mastoiditis. A 70-year-old male complained of fever and severe otorrhea from left external auditory canal 2 months after undergoing a craniotomy to remove a hematoma. He had right-sided paralysis and undertook bed rest. Brain computed tomography revealed continuous fluid accumulation in the left mastoid air cells and middle ear from left external auditory canal in addition to leukocytosis and increased C-reactive protein level. The tympanic membrane was severely swelling. These results indicated the presence of otitis media and mastoiditis. Otorrhea culture showed large amounts of M. slooffiae. The administration of liposomal amphotericin B (L-AMB), the irrigation of external auditory canal with normal saline, and the application of topical ketoconazole ointment were started. The administration of L-AMB for 8 weeks and voriconazole, which was switched from L-AMB, for 4 weeks ameliorated his infection and he was transferred to another hospital to receive rehabilitation. From these results and his clinical course, the diagnosis of otitis externa caused by Malassezia slooffiae complicated with mastoiditis was made. And the possibility of the contamination by M. slooffiae was very low. Clinicians should be aware that M.slooffiae can provoke otological infections since M. slooffiae is the most common Malassezia sp. in external auditory canal.

Sequential Treatment by Antiviral Drugs Followed by Immunosuppressive Agents for COVID-19 Patients with Hematological Malignancy.

Background: The detailed treatment regimen of COVID-19 patients with hematological malignancies has been unclear, and some fatalities have occurred, although combination therapy with antiviral agents and corticosteroids has been established for moderate to severe COVID-19 patients. Case Series: Case 1 was a 57-year-old woman who had malignant lymphoma and received CHOP therapy with obinutuzumab, and case 2 was a 70-year-old-man who had myeloma and received molecular targeted therapy with weekly corticosteroid. In both cases, SARS-CoV-2 genes and antigens were detected from their nasal swabs, and treatment was started for moderate to severe COVID-19. Case 1 received antiviral agents with high doses of corticosteroids for a long term simultaneously, but the high titer of viral antigens in her nasal swabs persisted. Ground-glass opacities and interstitial shadows also worsened in both lungs, and she finally died on day 60. In contrast, in case 2, antiviral agents were started first, and restarted the immunosuppressive agents, such as gamma globulin and corticosteroids after no titer of SARS-CoV-2 antigens was confirmed. The patient survived, and his abnormal chest shadows showed gradual improvement. Both of the patients received two vaccinations, but showed the low antibody titers for SARS-CoV-2. Conclusion: Administration of both antiviral agents and corticosteroids has been recommended for moderate to severe COVID-19 patients, but in patients with hematological malignancies, it might be better to use antiviral agents first to reduce the viral titers, and then add steroid and related immunosuppressive agents later appropriately to inhibit the excessive inflammatory state. The dose, timing, and order of the antivirals and immunosuppressive agents for COVID-19 should be considered carefully in the patients with hematological malignancies who showed low vaccine effectiveness.

A Mycobacterium tuberculosis-Infected Patient Who Could Not Tolerate Oral Intake Successfully Treated Using an Intravenous Tedizolid-Containing Regimen.

BACKGROUND Mycobacterium tuberculosis (M. tuberculosis) is usually treated by oral antimycobacterial agents, including rifampicin, ethambutol, and pyrazinamide, but the treatment regimen with intravenous and/or intramuscular antimycobacterial agents for patients who cannot take medications orally remains unclear. CASE REPORT A 77-year-old man with chronic renal failure had an esophageal-skin fistula after he had surgeries for removal of esophageal and gastric cancers and reconstruction using jejunum, and he showed a cavity, tree-in-bud formation, and pleural effusions in his left upper lung fields on his chest X-ray after treatment of cellulitis and bacteremia/candidemia by meropenem, teicoplanin, and micafungin. M. tuberculosis was isolated from his sputum and exudate fluid from the reconstructed esophageal-skin fistula. Although he could not take antimycobacterial agents orally, treatment was started with intravenous agents combining levofloxacin (LVFX) every other day, isoniazid (INH), and linezolid (LZD). However, his platelets were decreased 21 days after treatment started, and it was thought to be an adverse effect of LZD and/or INH. After changing LZD to tedizolid (TZD), in addition to changing from INH to intramuscular streptomycin twice per week, his platelet counts increased. Intravenous TZD could be continued, and it maintained his condition without exacerbations of thrombocytopenia and renal failure. The M. tuberculosis disappeared, and the abnormal chest X-ray shadows were improved 2 months after the start of treatment. CONCLUSIONS Administration of intravenous TZD, in addition to intravenous LVFX and intramuscular SM in combination, might be a candidate regimen for M. tuberculosis patients who cannot take oral medications.

Direct microorganism species identification and antimicrobial susceptibility tests from positive blood culture bottles using rapid Sepsityper Kit.

INTRODUCTION: We evaluated the performance of Rapid Sepsityper Kit in species identification (ID) and antimicrobial susceptibility testing (AST). METHODS: Positive blood culture bottles (n = 227) containing single microorganisms were enrolled. We compared the direct method using Rapid Sepsityper Kit for ID and AST with the conventional method. The analyses of ID and AST were performed using MALDI Biotyper and BD Phoenix platform, respectively. RESULTS: The direct ID method correctly identified 89.4% (203/227) of samples, and Gram-negative bacilli (95.2%) had a higher ID rate than Gram-positive cocci (84.4%). Five cases were misidentified, and non-acceptable identification was high among Streptococcus species. Direct AST results were obtained from 168 isolates. Non-acceptable ID occurred among 24 isolates; 4 Streptococcus species, and 31 isolates, which did not grow in the direct AST method, were excluded. A total of 1714 antibiotic susceptibility tests (625 from 69 Gram-positive cocci and 1089 from 99 Gram-negative bacilli) were performed. The direct AST methods showed 98.3% (1685/1714) of categorical agreement (CA), 0.7% (12/1714) of very major errors, 0.2% (4/1714) of major errors, and 0.8% (13/1714) of minor errors. Complete CA was obtained for methicillin-resistant Staphylococcus aureus and extended-spectrum beta-lactamase-producing Escherichia coli. CONCLUSIONS: The direct ID method using Rapid Sepsityper Kit and the direct AST method in combination with the BD Phoenix platform, which was associated with a reduction of turnaround time, may be a reliable approach for blood culture bottles. However, additional validation and further improvements, especially for Gram-positive cocci, would have an impact on microbiological diagnoses.

Infected aortic aneurysm caused by Streptococcus pyogenes: A case report.

We reported the case with infected abdominal aortic aneurysm (AAA) caused by Streptococcus (S.) pyogenes. A seventy-seven-year-old man, who had the history of uncontrolled diabetes mellitus (DM), complained fever and abdominal pain. Abdominal computed tomography scan revealed the aneurysm above common iliac artery with false lumen. On admission, laboratory tests found marked elevation of inflammatory biomarkers. Thereby the infected AAA was suspected and blood culture was taken. The administration of meropenem (MEPM) and daptomycin (DAP) was started. Next day he underwent abdominal aortic replacement with prosthetic graft and debridement because of persistent abdominal pain and the enlargement of aneurysm. S. pyogenes in blood culture samples was identified by Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry. Same result was obtained from the tissue samples of the resected AAA. Then the diagnosis of infected AAA caused by S. pyogenes was made. Since isolated S. pyogenes showed the susceptibility to antibiotics tested including penicillin, antibiotics were changed to ampicillin (ABPC) for the de-escalation of antibiotics. He had kept the administration of ABPC for 4 weeks and transferred to another hospital for the further treatment of DM. The aneurysms by S. pyogenes are extremely rare, but we should note that S. pyogenes could induce the aneurysms.

Thoracic aortic mycotic aneurysm due to Staphylococcus argenteus: A case report.

Staphylococcus argenteus was subdivided as a novel species from Staphylococcus aureus in 2014. We herein report a case of mycotic aneurysm caused by S. argenteus. A 59-year-old woman with diabetes and schizophrenia visited at the emergency room because of falling. Chest computed tomography revealed a left humerus fracture and a thoracic aortic aneurysm. With her elevated WBC count and CRP level, she was suspected to have a mycotic aneurysm. After being transferred to our hospital, vascular graft replacement surgery was performed. Isolates of blood cultures and surgical specimens were identified as S. argenteus by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MAS MALDI Biotyper Ver. 8.0). Although S. argenteus lacks staphyloxanthin, a carotenoid pigment, it is coagulase positive. In addition to traditional and automated biochemical identification systems, even MALDI-TOF MAS may misidentify the organism as S. aureus depending on its version. S. argenteus should be considered when coagulase-negative Staphylococcus like colonies are obtained from samples of S. aureus infection. To our knowledge, this is the first case of aortic mycotic aneurysm caused by S. argenteus in Japan. Although S. argenteus is considered less virulent than Staphylococcus aureus, we should closely monitor the prevalence and the clinical impact of this pathogen on community-acquired infections and health care-associated infections.

Antimicrobial susceptibility and prevalence of resistance genes in Bacteroides fragilis isolated from blood culture bottles in two tertiary care hospitals in Japan.

The in vitro susceptibilities of Bacteroides fragilis to antimicrobial agents, especially to carbapenem, are a major concern in the treatment of patients with bloodstream infections. In this study, 50 isolates of B. fragilis were obtained from positive blood bottles from 2014 to 2019 in Saitama, Japan. Their susceptibility to ampicillin/sulbactam was reduced to 70.0% compared with a previous report, whereas they were still sufficiently susceptible to piperacillin/tazobactam (94.0%). Five cfiA-positive isolates (5/50, 10.0%) were identified that were resistant to doripenem and meropenem, and two of them carried an insertion sequence located upstream of the cfiA-coding region. In particular, imipenem should be considered as a first-line carbapenem for the empirical treatment of B. fragilis infection because only insertion sequence and cfiA double-positive strains showed resistance to imipenem. Thirty-six percent of the isolates had a reduced minimum inhibitory concentration for moxifloxacin. In addition, metronidazole should still be considered as an active agent for B. fragilis because all isolates were susceptible to this antibiotic and the prevalence of the nim gene was low in Japan.

Rapid and Accurate Species Identification of Mitis Group Streptococci Using the MinION Nanopore Sequencer.

Differentiation between mitis group streptococci (MGS) bacteria in routine laboratory tests has become important for obtaining accurate epidemiological information on the characteristics of MGS and understanding their clinical significance. The most reliable method of MGS species identification is multilocus sequence analysis (MLSA) with seven house-keeping genes; however, because this method is time-consuming, it is deemed unsuitable for use in most clinical laboratories. In this study, we established a scheme for identifying 12 species of MGS (S. pneumoniae, S. pseudopneumoniae, S. mitis, S. oralis, S. peroris, S. infantis, S. australis, S. parasanguinis, S. sinensis, S. sanguinis, S. gordonii, and S. cristatus) using the MinION nanopore sequencer (Oxford Nanopore Technologies, Oxford, UK) with the taxonomic aligner "What's in My Pot?" (WIMP; Oxford Nanopore's cloud-based analysis platform) and Kraken2 pipeline with the custom database adjusted for MGS species identification. The identities of the species in reference genomes (n = 514), clinical isolates (n = 31), and reference strains (n = 4) were confirmed via MLSA. The nanopore simulation reads were generated from reference genomes, and the optimal cut-off values for MGS species identification were determined. For 31 clinical isolates (S. pneumoniae = 8, S. mitis = 17 and S. oralis = 6) and 4 reference strains (S. pneumoniae = 1, S. mitis = 1, S. oralis = 1, and S. pseudopneumoniae = 1), a sequence library was constructed via a Rapid Barcoding Sequencing Kit for multiplex and real-time MinION sequencing. The optimal cut-off values for the identification of MGS species for analysis by WIMP and Kraken2 pipeline were determined. The workflow using Kraken2 pipeline with a custom database identified all 12 species of MGS, and WIMP identified 8 MGS bacteria except S. infantis, S. australis, S. peroris, and S. sinensis. The results obtained by MinION with WIMP and Kraken2 pipeline were consistent with the MGS species identified by MLSA analysis. The practical advantage of whole genome analysis using the MinION nanopore sequencer is that it can aid in MGS surveillance. We concluded that MinION sequencing with the taxonomic aligner enables accurate MGS species identification and could contribute to further epidemiological surveys.

Bloodstream infection by multidrug-resistant Streptococcus oralis in a leukemic patient with febrile neutropenia after hematopoietic stem cell transplantation.

We reported the case of a patient with leukemia who developed febrile neutropenia after hematopoietic stem cell transplantation. Blood culture results revealed the presence of Streptococcus oralis, while antimicrobial susceptibility testing showed the resistance to penicillin and cephem. Furthermore, isolates were not susceptible to either meropenem or daptomycin but not to vancomycin. S oralis is known to belong to Streptococcus mitis group and be a causative agent of bacteremia in the neutropenic patients, but multidrug resistance of S oralis is rare. Our findings suggest that we might pay attention to the emergence of the microorganisms acquiring multidrug resistance in neutropenic patients.

Clinical characteristics in blood stream infections caused by Klebsiella pneumoniae, Klebsiella variicola, and Klebsiella quasipneumoniae: a comparative study, Japan, 2014-2017.

BACKGROUND: Klebsiella variicola and K. quasipneumoniae are new species distinguishable from K. pneumoniae but they are often misidentified as K. pneumoniae in clinical settings. Several reports have demonstrated the possibility that the virulence factors and clinical features differ among these three phylogroups. In this study, we aimed to clarify whether there were differences in clinical and bacterial features between the three phylogroups isolated from patients with bloodstream infections (BSIs) in Japan. METHODS: Isolates from all patients with BSIs caused by K. pneumoniae admitted to two hospitals between 2014 and 2017 (n = 119) were included in the study. Bacterial species were identified via sequence analysis, and their virulence factors and serotypes were analyzed via multiplex PCR results. Clinical data were retrieved from medical records. RESULTS: Of the 119 isolates, 21 (17.7%) were identified as K. variicola and 11 (9.2%) as K. quasipneumoniae; K1 serotype was found in 16 (13.4%), and K2 serotype in 13 (10.9%). Significant differences in the prevalence of rmpA, iutA, ybtS, entB and kfu (p < 0.001), and allS genes (p < 0.05) were found between the three phylogroups. However, there were no significant differences in clinical features, including the 30-day mortality rate, between the three organisms, although K. variicola was more frequently detected in patients over 80 years old compared with other Klebsiella species (p < 0.005), and K. quasipneumoniae more frequently occurred in patients with malignancy (p < 0.05). CONCLUSIONS: Our findings demonstrated the differences in bacterial pathogenicity and clinical features among these three phylogroups. Further epidemiological studies into BSI caused by Klebsiella species are warranted.

An identification protocol for ESBL-producing Gram-negative bacteria bloodstream infections using a MinION nanopore sequencer.

PURPOSE: The new third-generation sequencing platform MinION is an attractive maintenance-free and disposable portable tool that can perform long-read 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 were collected from patients with bloodstream infections, and 18 isolates of Gram-negative (GN) bacteria and 20 isolates of Gram-positive (GP) bacteria were identified from these using 16S rRNA sequencing and then used in this study. 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 identifications made by MinION sequencing for 30 min using the What's In My Pot? (WIMP) workflow via EPI2ME on the basis of the most frequent classified reads were consistent with those made by 16S rRNA sequencing. On the other hand, for GP bacteria specimens, the identification results for 16S rRNA sequencing and MinION were only in agreement in 12 out of 20 (60.0 %) cases. ARMA analysis was able to detect extended-spectrum ß-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 the confirmation of an ESBL phenotype. This innovative sequence technology and its application could lead to a breakthrough in the diagnosis of infectious diseases.