A case of hypervirulent K1-ST23 Klebsiella pneumoniae endocarditis and papillary muscle rupture secondary to multiple site abscesses.

Hypervirulent Klebsiella pneumoniae (hvKP) causes multisite infections and abscesses. However, endocarditis is a rare presentation of hvKP infection. Herein, we report a case of K. pneumoniae native valve infective endocarditis secondary to community-acquired liver and prostate abscesses. The patient developed papillary muscle rupture, leading to mitral regurgitation, and underwent emergent mitral valve replacement. The diagnosis of endocarditis was confirmed microbiologically and histologically. The causative strain belonged to the hypermucoid K1 capsular genotype and possessed the rmpA gene. The genome sequence was deposited in GenBank under the accession number JAQZBZ000000000.

Human abdominal abscess caused by Necropsobacter rosorum and tips for its identification: A case report.

Necropsobacter rosorum is a gram-negative facultative anaerobe, which was reclassified from the family Pasteurellaceae in 2011. It has been detected in the gastrointestinal and respiratory tracts of mammals; however, reports of infection in humans are scarce. We report a case of an abdominal abscess in which N. rosorum was detected; it was successfully treated with drainage and antimicrobial therapy. Routine laboratory testing such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and an identification system using biochemical phenotypes could not identify N. rosorum. Instead, it was misidentified as other Pasteurellaceae species, including Aggregatibacter spp. or Pasteurella spp. Sequencing of 16S rRNA was required to identify N. rosorum. We suggest the application of simple methods, such as indole production, oxidase, and catalase tests, to differentiate N. rosorum from genetically similar species.

Tenosynovitis caused by Mycobacterium marseillense, initially identified as Mycobacterium avium complex using AccuProbe and COBAS TaqMan.

BACKGROUND: Mycobacterium marseillense is a new species of the Mycobacterium avium complex. There has been only a few human infections caused by M. marseillense worldwide. CASE PRESENTATION: We report a case of tenosynovitis caused by M. marseillense in an immunocompetent adult in Japan. The isolate was initially identified as M. intracellulare using commercial real time polymerase chain reaction assays and later identified as M. marseillense with sequencing of the the rpoB and hsp65 regions, and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). CONCLUSIONS: This is the first case reporting on M. marseillense generating a positive result with commercial real time PCR assays targeting MAC. Human infections associated by M. marseillense might be underreported due to similarities with Mycobacterium intracellulare. To accurately identify M. marseillese, MALDI-TOF MS might provide a rapid and reliable method.

Kluyvera georgiana Bacteremia Due to Acute Cholangitis: A Report of the First Known Case and a Literature Review.

We herein present the first known case of bacteremia caused by Kluyvera georgiana in a 67-year-old female undergoing chemotherapy for recurrent pancreatic cancer. The patient underwent choledochojejunotomy and thereafter developed ascending cholangitis. The diagnosis of K. georgiana was confirmed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. A literature review of Kluyvera spp. infections indicated potential risk factors including an underlying malignancy and immunosuppression. Although Kluyvera spp. infections are typically sensitive to antibiotics, multidrug resistance is possible. This case highlights the importance of the early diagnosis and treatment of K. georgiana and its associated risk factors.

Two cases of bacteremia caused by Leptotrichia trevisanii in patients with febrile neutropenia.

We present two cases of bacteremia caused by Leptotrichia trevisanii: a 12-year-old girl with recurrent myeloid leukemia of the mandible and a 66-year-old man with esophageal carcinoma. As this filamentous bacillus showed indefinite Gram staining and the identification based on biochemical enzymatic reactions was not definitive, identification required 16s rRNA analysis. For this organism, drug sensitivity testing showed susceptiblity to each ß-lactam antibiotics and clindamycin, but resistance to fluoroquinolone and erythromycin. This filamentous bacillus needs careful identification and appropriate antibiotic treatment.

Comparison of phylogenetic and virulence factors between Escherichia coli isolated from biliary tract infections and uropathogenic Escherichia coli.

Escherichia coli is a gram-negative intestinal commensal that can also cause various infections, including urinary tract infections, biliary tract infections, neonatal meningitis, and septicemia. Although the characteristics of uropathogenic E. coli and the mechanisms of urinary tract infection have been well studied, the genetic distinctions among E. coli isolates from different types of infections have not yet been determined. This study compared the phylogenetic and virulence factors of E. coli isolates from bacteremic biliary tract infections with those from bacteremic urinary tract infections. The phylogenetic B2 group was the most prevalent in both pathogenic groups (68 % in biliary pathogenic isolates and 85 % in uropathogenic isolates), but the frequency pattern of the phylogenetic group was different. Half of the uropathogenic isolates belonged to ST95 and ST131 (51 %). Among the biliary pathogenic isolates, ST131 was the most prevalent, while the remaining half belonged to other STs outside the four major STs. The frequency of some virulence factors, such as papC, papG2, hlyA, tcpC, fyuA, kpsMT2, sat, and traT, was lower in the biliary pathogenic isolates than in the uropathogenic isolates. The frequency of phylogenetic groups and STs in MLST differed between E. coli isolates from bacteremic biliary tract infections and urinary tract infections. Additionally, some virulence factors, including adhesion and toxin gene groups, showed lower frequencies in the biliary pathogenic group than in the uropathogenic group. Studying the differences in E. coli pathovars from different infection sites is important for developing pathovar-specific targeted therapies such as vaccine therapy.

Accurate Identification of Klebsiella variicola by MALDI-TOF Mass Spectrometry in Clinical Microbiology Laboratories.

Klebsiella variicola is a pathogen that is increasingly recognized as being associated with human infections, but the methods available to clinical microbiology laboratories for accurate identification are limited. In this study, we assessed the accuracy of identification of K. variicola by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry using genetic identification with multiplex PCR as the reference method. Antimicrobial susceptibilities and virulence of K. variicola strains were also investigated. Fifty-five Klebsiella pneumoniae, 26 K. variicola, and 2 Klebsiella quasipneumoniae clinical strains were used for evaluation. Both MALDI Biotyper with library version 9 and Klebsiella MALDI TypeR, a web-based species identification tool using MALDI-TOF data, accurately identified all K. variicola strains. In addition, two strains of K. quasipneumoniae were accurately identified with Klebsiella MALDI TypeR. Whole-genome sequencing confirmed the accurate identification to the subspecies level by Klebsiella MALDI TypeR for four strains (two strains each of K. variicola subsp. variicola and K. quasipneumoniae subsp. similipneumoniae). While 13 strains, 3 strains, and 1 strain of K. pneumoniae showed nonsusceptibility to ampicillin-sulbactam, ceftriaxone, and meropenem, respectively, all strains of K. variicola were susceptible to all tested antimicrobial agents. Although two K. variicola strains were positive for the string test, no K. variicola strains harbored any of the genes associated with hypervirulence of K. pneumoniae. Accurate identification of the K. pneumoniae complex, including K. variicola, by MALDI-TOF in clinical microbiology laboratories is expected to clarify the clinical characteristics of each species in the future. IMPORTANCE Recent widespread use of bacterial whole-genome sequencing analysis has resulted in the proposal of novel bacterial species and reclassification of taxonomy. Accurate methods for identification of bacterial species in clinical microbiology laboratories are essential to accumulate information on the clinical characteristics of each bacterial species. Klebsiella variicola is a member of the Klebsiella pneumoniae complex, and its association with human infections has been increasingly recognized, but accurate identification methods approved for use in clinical microbiology laboratories have been limited thus far. The findings of the present study suggest that K. variicola can be accurately identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry using updated library or web-based identification tools. Accurate identification will promote exploration of clinical characteristics of K. variicola.

Comparative performance and cycle threshold values of 10 nucleic acid amplification tests for SARS-CoV-2 on clinical samples.

BACKGROUND: A number of nucleic acid amplification tests (NAATs) for SARS-CoV-2 with different reagents have been approved for clinical use in Japan. These include research kits approved under emergency use authorization through simplified process to stabilize the supply of the reagents. Although these research kits have been increasingly used in clinical practice, limited data is available for the diagnostic performance in clinical settings. METHODS: We compared sensitivity, specificity, and cycle threshold (Ct) values obtained by NAATs using 10 kits approved in Japan including eight kits those receiving emergency use authorization using 69 frozen-stored clinical samples including 23 positive samples with various Ct values and 46 negative samples. RESULTS: Viral copy number of the frozen-stored samples determined with LightMix E-gene test ranged from 0.6 to 84521.1 copies/µL. While no false-positive results were obtained by any of these tests (specificity: 100% [95% CI, 88.9%-100%]), sensitivity of the nine tests ranged from 68.2% [95% CI, 45.1%-86.1%] to 95.5% [95% CI, 77.2%-99.9%] using LightMix E-gene test as the gold standard. All tests showed positive results for all samples with ≥100 copies/µL. Significant difference of Ct values even among tests amplifying the same genetic region (N1-CDC, N2) was also observed. CONCLUSION: Difference in the diagnostic performance was observed among NAATs approved in Japan. Regarding diagnostic kits for emerging infectious diseases, a system is needed to ensure both rapidity of reagent supply and accuracy of diagnosis. Ct values, which are sometimes regarded as a marker of infectivity, are not interchangeable when obtained by different assays.

Clinical and Microbiological Characteristics of Recurrent Escherichia coli Bacteremia.

The causative agents of recurrent Escherichia coli bacteremia can be genetically identical or discordant, but the differences between them remain unclear. This study aimed to explore these differences, with regard to their clinical and microbiological features. Patients were recruited from a Japanese tertiary teaching hospital based on blood culture data and the incidence of recurrent E. coli bacteremia. We compared the patients' clinical and microbiological characteristics between the two groups (those with identical or discordant E. coli bacteremia) divided by the result of enterobacterial repetitive intergenic consensus PCR. Among 70 pairs of recurrent E. coli bacteremia strains, 49 pairs (70%) were genetically identical. Patients with genetically identical or discordant E. coli bacteremia were more likely to have renal failure or neoplasms, respectively. The virulence factor (VF) scores of genetically identical E. coli strains were significantly higher than those of genetically discordant strains, with the prevalence of eight VF genes being significantly higher in genetically identical E. coli strains. No significant differences were found between the two groups regarding antimicrobial susceptibility and biofilm formation potential. This study showed that genetically identical E. coli bacteremia strains have more VF genes than genetically discordant strains in recurrent E. coli bacteremia. IMPORTANCE Escherichia coli causes bloodstream infection, although not all strains are pathogenic to humans. In some cases, this infection reoccurs, and several reports have described the clinical characteristics and/or molecular microbiology of recurrent Escherichia coli bacteremia. However, these studies focused on patients with specific characteristics, and they included cases caused by microorganisms other than Escherichia coli. Hence, little is known about the pathogenicity of Escherichia coli isolated from the recurrent one. The significance of our study is in evaluating the largest cohorts to date, as no cohort studies have been conducted on this topic.

The prevalence of the iutA and ibeA genes in Escherichia coli isolates from severe and non-severe patients with bacteremic acute biliary tract infection is significantly different.

BACKGROUND: Although Escherichia coli is the most frequently isolated microorganism in acute biliary tract infections with bacteremia, data regarding its virulence are limited. RESULTS: Information on cases of bacteremia in acute biliary tract infection in a retrospective study was collected from 2013 to 2015 at a tertiary care hospital in Japan. Factors related to the severity of infection were investigated, including patient background, phylogenetic typing, and virulence factors of E. coli, such as adhesion, invasion, toxins, and iron acquisition. In total, 72 E. coli strains were identified in 71 cases, most of which primarily belonged to the B2 phylogroup (68.1%). The presence of the iutA gene (77.3% in the non-severe group, 46.4% in the severe group, P = 0.011) and the ibeA gene (9.1% in the non-severe group, and 35.7% in the severe group, P = 0.012) was significantly associated with the severity of infection. Among the patient characteristics, diabetes mellitus with organ involvement and alkaline phosphatase were different in the severe and non-severe groups. CONCLUSIONS: We showed that bacteremic E. coli strains from acute biliary tract infections belonged to the virulent (B2) phylogroup. The prevalence of the iutA and ibeA genes between the two groups of bacteremia severity was significantly different.

First Reported Human Case of Spondylodiscitis by Staphylococcus condimenti: A Case Report and Literature Review.

Staphylococcus condimenti is a Gram-positive coccus that was first isolated from soy sauce mash. Only four cases of human S. condimenti infections have been reported to date. We herein report the first case of spondylodiscitis caused by S. condimenti. A 72-year-old Japanese man complaining of lower back pain and numbness in his legs was diagnosed with spondylodiscitis. A computed tomography (CT)-guided biopsy was performed. A culture of the intravertebral disc aspirate yielded S. condimenti. The result was confirmed using gene sequencing methods. The patient was successfully treated without relapse. This case shows that S. condimenti can be pathogenic and cause invasive infection.

Association of the Serum Levels of the Nucleocapsid Antigen of SARS-CoV-2 With the Diagnosis, Disease Severity, and Antibody Titers in Patients With COVID-19: A Retrospective Cross-Sectional Study.

Background: Several types of laboratory tests for COVID-19 have been established to date; however, the clinical significance of the serum SARS-CoV-2 nucleocapsid (N) antigen levels remains to be fully elucidated. In the present study, we attempted to elucidate the usefulness and clinical significance of the serum N antigen levels. Methods: We measured the serum N antigen levels in 391 serum samples collected from symptomatic patients with a confirmed diagnosis of COVID-19 and 96 serum samples collected from patients with non-COVID-19, using a fully automated chemiluminescence immunoassay analyzer. Results: Receiver operating characteristic analysis identified the optimal cutoff value of the serum N antigen level (cutoff index, based on Youden's index) as 0.255, which yielded a sensitivity and specificity for the diagnosis of COVID-19 of 91.0 and 81.3%, respectively. The serum N antigen levels were significantly higher in the patient groups with moderate and severe COVID-19 than with mild disease. Moreover, a significant negative correlation was observed between the serum N antigen levels and the SARS-CoV-2 IgG antibody titers, especially in patients with severe COVID-19. Conclusion: Serum N antigen testing might be useful both for the diagnosis of COVID-19 and for obtaining a better understanding of the clinical features of the disease.

Genetic diversity and epidemiology of accessory gene regulator loci in Clostridioides difficile.

Quorum sensing is known to regulate bacterial virulence, and the accessory gene regulator (agr) loci is one of the genetic loci responsible for its regulation. Recent reports examining Clostridioides difficile show that two agr loci, agr1 and agr2, regulate toxin production, but the diversity of agr loci and their epidemiology is unknown. In our study, in silico analysis was performed to research genetic diversity of agr, and C. difficile isolates from clinical samples underwent multilocus sequence typing (MLST) and PCR analysis of agr loci. To reveal the distribution of agr among different strains, phylogenetic analysis was also performed. In our in silico analysis, two different subtypes, named agr2R and agr2M, were found in agr2, which were previously reported. PCR analysis of 133 C . difficile isolates showed that 131 strains had agr1, 61 strains had agr2R, and 26 strains had agr2M; agr2R was mainly found in clade 1 or clade 2 organisms, whereas agr2M was only found in clade 4. With rare exception, agr1-negative sequence types (STs) belonged to clade C-Ⅰ and C-Ⅲ, and one clade 4 strain had agr2R. Our study revealed subtypes of agr2 not previously recognized, and the distribution of several agr loci in C. difficile . These findings provide a foundation for further functional and clinical research of the agr loci.

Epidemiology and virulence-associated genes of Clostridioides difficile isolates and factors associated with toxin EIA results at a university hospital in Japan.

INTRODUCTION: Clostridioides difficile is one of the most important nosocomial pathogens; however, reports regarding its clinical and molecular characteristics from Japan are scarce. AIMS: We studied the multilocus sequence typing (MLST)-based epidemiology and virulence-associated genes of isolates and the clinical backgrounds of patients from whom the isolates had been recovered. METHODS: A total of 105 stool samples tested in a C. difficile toxin enzyme immune assay (EIA) were analysed at the University of Tokyo Hospital from March 2013 to July 2014. PCR for MLST and the virulence-associated genes tcdA, tcdB, cdtA, cdtB and tcdC was performed on C. difficile isolates meeting our inclusion criteria following retrospective review of medical records. EIA-positive and EIA-negative groups with toxigenic strains underwent clinical and molecular background comparison. RESULTS: The toxigenic strains ST17, ST81, ST2, ST54, ST8, ST3, ST37 and ST53 and the non-toxigenic strains ST109, ST15 and ST100 were frequently recovered. The prevalence rate of tcdA-negative ST81 and ST37, endemic in China and Korea, was higher (11.4%) than that reported in North America and Europe, and hypervirulent ST1(RT027) and ST11(RT078) strains that occur in North America and Europe were not recovered. The linkage between the EIA results and cdt A/B positivity, tcdC deletion, or tcdA variation was absent among toxigenic strains. Compared with the 38 EIA-negative cases, the 36 EIA-positive cases showed that the patients in EIA-positive cases were older and more frequently had chronic kidney disease, as well as a history of beta-lactam use and proton pump inhibitor therapy. CONCLUSION: In Japan, the prevalence rates for tcdA-negative strains are high, whereas the cdtA/B-positive strains are rare. EIA positivity is linked to older age, chronic kidney disease and the use of beta-lactams and proton pump inhibitors.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry can be used to identify Helicobacter cinaedi.

We examined the applicability of Matrix-assisted laser desorption ionization-time of flight mass spectrometry using 54 Helicobacter cinaedi isolates from humans. In all 54 isolates, MALDI-TOF MS detected H. cinaedi as the best match organism. Our findings suggest that MALDI TOF-MS can be used effectively to identify H. cinaedi.

[The fundamental evaluation of COBAS TaqMan48 using clinical specimens].

PURPOSE: When smear test is positive for acid-fast bacilli, it is important to differentiate between Mycobacterium tuberculosis complex and nontuberculosis mycobacteria (NTM) in healthcare associated infection (HAI) control. The aim of this study is to evaluate between COBAS TaqMan48 (TaqMan) and COBAS Amplicor (COBAS). MATERIAL AND METHOD: Tenfold dilution series of 5 x 10(4) cfu/mL of Mycobacterium bovis (ATCC19210) were used for evaluating the limit of detection (LOD) and reproducibility. 73 frozen clinical specimens (34 M. tuberculosis complex and 39 NTM) stored at below -20 degrees C before its use that were treated with NALC-NaOH were used to determine the agreement between two methods. Divided reaction reagents (include Master mix solution, Internal control and Mg2+) of TaqMan for 3 and 6 tests per batch used to evaluate reagents stability. RESULT: The LOD of both kits were 5 X102 cfu/mL. Regarding reproducibility, the same result was obtained when tested 3 times. The agreement rate between TaqMan and culture method was 58.8%, and between COBAS and culture method was 67.6%. When limited to smear positive eleven specimens, the agreement between TaqMan and culture method was 81.8%, and between COBAS and culture method was 90.9%. Reagents divided for 3 tests and 6 tests and stored at 4 degrees C in dark, both test reagents stability was confirmed maximum for 16 days. CONCLUSION: As the results of the evaluation of TaqMan, the LOD, reproducibility and the agreement were similar to COBAS results. However, in low colony forming unit of clinical specimen raise the possibility that results may contain false negative.

Evaluation of a simple phenotypic method for the detection of carbapenemase-producing Enterobacteriaceae.

We investigated the performance of a phenotypic test, the Carbapenemase Detection Set (MAST-CDS), for the identification of carbapenemase-producing Enterobacteriaceae. Our results indicated that MAST-CDS is rapid, easily performed, simple to interpret, and highly sensitive for the identification of carbapenemase producers, particularly imipenemase producers.

Antibiotic Susceptibilities of Pseudomonas aeruginosa Isolated from Blood Samples and Antibiotic Utilization in a University Hospital in Japan.

INTRODUCTION: Pseudomonas aeruginosa is one of the most important causes of nosocomial infection. Several reports indicated a correlation of antimicrobial usages and declined susceptibilities. In this report, we evaluated their relation in a tertiary care teaching hospital in Tokyo, Japan for 4 years. METHODS: We evaluated the susceptibilities of 149 strains of P. aeruginosa isolated from blood samples and consumption of anti-pseudomonal antibiotics as antimicrobial use density from 2009 to 2012 in the University of Tokyo Hospital in Tokyo, Japan. RESULTS: Usages of carbapenems and anti-pseudomonal cephalosporins decreased 44% and 31% from 2009 to 2011, and then increased 30% and 24% in 2012, respectively. Usage of piperacillin-tazobactam increased 87% from 2009 to 2012, which was introduced in the hospital in 2008. Consumption of fluoroquinolones and aminoglycoside remained low in those years. Susceptibilities to cephalosporins, carbapenems (except for panipenem-betamipron), penicillins, and fluoroquinolones declined between 22% and 39% in 2010, increased in the range of 16-31% in 2011, and increased by 1-14% in 2012. Susceptibility of panipenem-betamipron ranged between 25% and 32%. Susceptibility to aminoglycoside was more than 90% during this period. No relationship between antimicrobial usages and susceptibilities of P. aeruginosa was observed. CONCLUSION: Susceptibilities of P. aeruginosa did not correlate with the usage of antibiotics in our hospital. Several infection control measures and other factors might contribute to changing the susceptibilities of bacteria.

[Reliability of Amplicor Mycobacteria Kit for detection of Mycobacterium tuberculosis complex and Mycobacterium intracellulare: results of cooperative study among 331 laboratories in 2000].

Amplicor Mycobacterium Kit (Roch Diagnostics: Japan) is the most widely used kit in Japan for the diagnosis of mycobacteria infections, especially those caused by Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium intracellulare. We evaluated the reliability of the kit in co-operation with 331 laboratories using the kit in routine examination. We distributed specially prepared 4 test samples to each laboratories. The negative sample was NALC-NaOH treated sputum which showed "negative" when tested by this kit and positive samples were NALC-NaOH treated sputum containing M. bovis or M. intracellurare. False-positive results were reported in 6 out of 331 laboratories (1.8%) and false-negative results were reported from 7 laboratories (2.1%). (The details were 1 out of 331 labs for TB-H sample, 5 out of 331 labs for TB-L sample and 1 out of 316 in MIN sample.) Statistical significance between MWP method and COBAS method was not significant. After receiving and evaluating the test results on the 4 samples, the follow up questionnaires were sent out to 22 laboratories which reported incorrect results and low optical density (O.D.) on positive control. Results of this questionnaire suggested that it was important to follow the package insert instructions and to follow the correct procedures for PCR assay. These results suggested that Amplicor Mycobacterium Kit is reliable for rapid diagnosis of Mycobacteria infections.

Pyogenic discitis due to Abiotrophia adiacens.

INTRODUCTION: Abiotrophia species have been referred to as nutritionally variant streptococci because of their fastidious nutritional requirements for growth. Abiotrophia species are difficult to identify with conventional solid culture. PRESENTATION OF CASE: A 48-year-old woman was admitted to our hospital with severe low back pain and body temperature of 38.2°C. Magnetic resonance imaging revealed edema and contrast enhancement of the L4 and L5 vertebral bodies with high signal intensity in the L3-4 and L4-5 intervertebral discs on the T2-weighted images. The patient underwent needle biopsy of the L3-4 disk. Cultures of disk biopsy samples and blood yielded gram positive cocci in short chains with scanty growth on chocolate agar. Further subculture with supplemented medium and subsequent 16S ribosomal RNA gene sequencing identified the pathogen as Abiotrhophia adiacens. The patient was treated with intravenous ampicillin. At 6-month follow-up, the patient was free of symptoms. DISCUSSION: Causative microorganisms remain unidentified in 25-40% of spinal infection cases. Abiotrophia species grow poorly on conventional solid media, and require pyridoxal or thiol group supplementation. Use of Brucella HK agar or GAM agar plate is helpful for detection of Abiotrophia species. We first confirmed the diagnosis by direct identification of Abiotrophia adiacens from infected disk. Abiotrophia species are one of the major pathogens of infective endocarditis accounting for 5% of cases. Considering their fastidious nature, it is likely that most cases of Abiotrophia discitis are falsely classified as culture-negative discitis; therefore, their role in pyogenic discitis may be underestimated. CONCLUSION: Subculture using nutritionally supplemented media is crucial for their identification.