Case of Chryseobacterium hominis Isolated from Human Blood Drawn Through Peripherally Inserted Central Catheter

Chryseobacterium hominis is non-fermenting Gram-negative rod that was first identified as a novel species in 2007. Here, we report the first clinical case of C. hominis bacteremia, which was confirmed by MALDI-TOF MS and 16S rRNA gene sequencing. A 16-year-old boy diagnosed with acute lymphoblastic leukemia was hospitalized for three months. Two sets of blood culture test through a peripherally inserted central catheter (PICC), which was inserted a month ago, was performed when his white blood cell count declined and he had a high fever. Colonies of medium sizes that looked round, mucoid, sticky, and grayish on blood and chocolate agar plates were observed. Identification of bacteria using the VITEK MALDI-TOF MS system (BioMérieux, France) was not successful and the VITEK 2 system (BioMérieux, USA) indicated Sphingomonas paucimobilis, with a questionable level of confidence (92%). However, Microflex LT Biotyper (Bruker Daltonics, Germany) showed C. homins (log score: 1.81) and sequence of 16S rRNA showed a 100% identity with C. hominis. Piperacillin-tazobactam was administered since the isolate was susceptible to piperacillin-tazobactam but C. hominis showed growth in the next four follow-up culture of blood drawn through PICC. The fever subsided only after PICC was changed. The clinical prognosis and antimicrobial susceptibility test of C. hominis should be further studied.ope

Urinary tract infection caused by a small colony variant form of capnophilic Escherichia coli leading to misidentification and non-reactions in antimicrobial susceptibility tests

Background: Small colony and capnophilic variant cases have been separately reported, but there has been no reports of their simultaneous presence in one isolate. We report a case of Escherichia coli with coexpressed small colony and capnophilic phenotypes causing misidentification in automated biochemical kits and non-reactions in antimicrobial susceptibility test cards. Case presentation: An 86-year-old woman developed urinary tract infection from a strain of Escherichia coli with SCV and capnophilic phenotypes in co-existence. This strain did not grow without the presence of CO2, and therefore proper identification from automated system was not possible. 16 s rRNA sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was able to identify the bacteria. Conclusion: As these strains do not grow on culture parameters defined by CLSI or on automated systems, proper identification using alternative methods are necessary.ope

Adjustment of Modified Carbapenem Inactivation Method Conditions for Rapid Detection of Carbapenemase-Producing Acinetobacter baumannii

Background: The existing modified carbapenem inactivation methods (mCIMs) recommended by the CLSI for detecting carbapenemase production have not been applicable for Acinetobacter baumannii. We evaluated the influence of matrices used in mCIMs and CIMTris on the stability of the disks for detecting carbapenemase producers and suggested optimal mCIM conditions for detecting carbapenemase-producing A. baumannii. Methods: Seventy-three A. baumannii isolates characterized for antimicrobial susceptibility and carbapenemase encoding genes were tested for carbapenemase production using mCIM and CIMTris. The influence of the matrices (Tryptic soy broth [TSB] and Tris-HCl) used in these methods on the stability of the meropenem (MEM) disk was also evaluated. The mCIM conditions were adjusted to enhance screening sensitivity and specificity for detecting carbapenemase-producing A. baumannii. Results: The matrices had an impact on the stability of the MEM disk after the incubation period (two or four hrs). TSB nutrient broth is an appropriate matrix for mCIM compared with Tris-HCl pH 7.6, which leads to the loss of MEM activity in CIMTris. The sensitivity and the specificity of the optimal mCIM were both 100%. Conclusions: We established optimal mCIM conditions for simple, accurate, and reproducible detection of carbapenemase-producing A. baumannii.ope

Determination of Colistin Resistance by Simple Disk Diffusion Test Using Modified Mueller-Hinton Agar

Background: Colistin has become a last-resort antibiotic for the management of multidrug-resistant gram-negative bacteria. The disk diffusion test is cheap and easy to perform but may be unreliable for colistin susceptibility testing due to poor diffusion of the large colistin molecule. An improved agar diffusion test would increase the reliability of colistin susceptibility testing. This study aimed to modify Muller-Hinton agar (MHA) to improve colistin diffusion in agar. Methods: MHA was modified by reducing the agar concentration from 100% to 30% and supplementing with protamine. We tested 60 gram-negative clinical isolates of Pseudomonas aeruginosa (N=27) and Acinetobacter calcoaceticus-baumannii complex (N=33). Disk diffusion test results were interpreted based on minimum inhibitory concentrations determined by broth microdilution. Results: The modified MHA yielded the best performance metrics, including 94.7% sensitivity, 100% specificity, and an area under the curve of 0.995 (95% confidence interval, 0.982-1.000), P<0.001, at a cut-off point of 13 mm. Conclusions: A reduction of the agar concentration from 100% to 30% and the addition of protamine improved colistin diffusion in agar and allowed routine colistin susceptibility testing in a clinical microbiology laboratory, but should be handled with caution.ope

Septicemia Caused by Herbaspirillum huttiense Secondary to Pneumonia

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Application of the Whole Genome-Based Bacterial Identification System, TrueBac ID, Using Clinical Isolates That Were Not Identified With Three Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) Systems

BACKGROUND: Next-generation sequencing is increasingly used for taxonomic identification of pathogenic bacterial isolates. We evaluated the performance of a newly introduced whole genome-based bacterial identification system, TrueBac ID (ChunLab Inc., Seoul, Korea), using clinical isolates that were not identified by three matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) systems and 16S rRNA gene sequencing. METHODS: Thirty-six bacterial isolates were selected from a university-affiliated hospital and a commercial clinical laboratory. Species was identified by three MALDI-TOF MS systems: Bruker Biotyper MS (Bruker Daltonics, Billerica, MA, USA), VITEK MS (bioMérieux, Marcy l'Étoile, France), and ASTA MicroIDSys (ASTA Inc., Suwon, Korea). Whole genome sequencing was conducted using the Illumina MiSeq system (Illumina, San Diego, CA, USA), and genome-based identification was performed using the TrueBac ID cloud system (www.truebacid.com). RESULTS: TrueBac ID assigned 94% (34/36) of the isolates to known (N=25) or novel (N=4) species, genomospecies (N=3), or species group (N=2). The remaining two were identified at the genus level. CONCLUSIONS: TrueBac ID successfully identified the majority of isolates that MALDI-TOF MS failed to identify. Genome-based identification can be a useful tool in clinical laboratories, with its superior accuracy and database-driven operations.ope

Performance of Microflex LT Biotyper and VITEK MS for Routine Identification of Yeasts.

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Fusobacterium nucleatum in biopsied tissues from colorectal cancer patients and alcohol consumption in Korea

The roles of individual bacteria and their relationship in the development of colorectal cancer (CRC) remain unclear. We aimed to determine the prevalence of CRC-associated bacteria using quantitative real-time PCR (qPCR) or 16S rRNA analysis and the statistical correlations of patient demographics and clinical characteristics comprising alcohol consumption with CRC-associated bacteria. We determined the prevalence of five CRC-associated bacterial species in 38 CRC patients (39 samples) and 21 normal individuals using qPCR, and the relative abundance of bacterial taxa in the gut microbiome was assessed using 16S rRNA analysis. Fusobacterium nucleatum was the only bacterium that was significantly (P < 0.0001) more prevalent in the cancer tissue (82.1%) than in the normal tissue (0%) by qPCR. 16S rRNA analysis showed a significant correlation between six operational taxonomic units (OTUs), namely, the genera Fusobacterium, Peptostreptococcus, Collinsella, Prevotella, Parvimonas, and Gemella, in patients with CRC. An integrated analysis using 16S rRNA data and epidemiological characteristics showed that alcohol consumption was significantly correlated with the abundance of Fusobacterium OTUs. The correlation of alcohol consumption with the abundance of Fusobacterium OTUs in cancer tissue discovered using 16S rRNA analysis suggests a possible link between alcohol metabolism and subsequent tumorigenesis caused by F. nucleatum.ope

약물의 대사 안정성과 형질을 평가하는 체계 구축

학위논문 (석사)-- 서울대학교 대학원 : 약학과, 2013. 2. 정석재.New chemical entity의 대사적 특성을 결정하는 것은 신약개발에서 중요한 과정이다. 이 중 CYP 효소 계에 의한 대사를 평가하는 대표적인 in vitro 실험법으로 metabolic stability와 phenotype 연구방법이 있다. 본 연구에서는 human liver microsomes (HLM)을 이용하여 metabolic stability와 phenotyping study를 평가하는 시스템을 본 연구실에서 구축한 뒤 이 방법에 따라 여러 물질의 stability를 평가하여 human in vivo CL 예측 성이 어떠한 지 밝히고자 하였다. 먼저 metabolic stability 평가 법을 검증하기 위해 잘 알려진 CYP의 기질들을 선정하고 HLM에서의 시간에 따른 잔존 약물의 양을 HPLC/UV를 통해 측정한 다음 동일한 약물에 대해 반복 실험하여 대사속도상수 ke 및 CLint를 구하였다. 그리고 구조적으로 다양한 31종의 약물에 대하여 추정 CL를 계산하고 실측 CL값과 비교하였다. 또한 CYP 기질과 경로 별 저해제를 동일 기질에 대하여 반복 실험하여 phenotype 평가 법을 구축하였다. 구축된 방법에 따라 신약으로 개발 중인 NDC002 물질에 대한 phenotype을 평가하였다. 31종의 약물의 in vitro에서 구한 추정 CL과 실측 CL 간에는 대체적으로 낮은 상관관계를 보였다. 또한, NDC002 약물은 주로 3A4를 통해 대사 되는 것으로 파악되었다. 본 연구를 통해 metabolic stability와 phenotype을 적절히 평가하는 연구방법이 개발되었으며 검증되었다. In vitro에서 microsomal stability 평가만으로는 in vivo CL을 정확히 예측하기에는 한계점이 있는 것을 알았으며 추가 소실경로의 확인 및 대사 소실을 제한하는 간으로의 약물 분포 특성 등을 동시에 고려해야 할 것으로 추정되었다. 따라서, microsomal stability 결과 만으로는 신약후보물질을 탈락시키는 것은 좋은 개발전략이 아닌 것으로 판단되었다.국문초록 2 목차 4 List of Tables 6 List of Figures 7 List of Abbreviations 14 1. Introduction 15 1.1.Drug development process and in vitro studies 15 1.2.Drug metabolism and CYP 18 2. Objectives 20 3. Material 21 3.1.Materials 21 3.2.Experimental instrument 22 4. Methods 23 4.1.Validation of metabolic stability 23 4.1.1.Incubations in microsomes 23 4.1.2.Standard solution 24 4.1.3.HPLC analysis 25 4.1.4.Data analysis 25 4.2.Estimation of in vivo CL from in vitro CL 29 4.3.Validation of phenotyping study 34 4.3.1.Incubations in microsome 34 4.3.2.Data analysis 35 4.4.Phenotyping study of unknown compound 39 5. Results 41 5.1.Validation of metabolic stability 41 5.2.Prediction of in vivo CL from in vitro CL 52 5.3.Validation of phenotyping study 73 5.4.Phenotyping test of K002 compound 81 6. Discussion 89Maste