Sang Hoon Song

A rapid antimicrobial susceptibility test based on single-cell morphological analysis

An antimicrobial susceptibility test rapidly identifies bacterial response to drugs based on imaged morphology of single cells. Microbes Get in Shape for Antibiotic Testing Conventional tests that measure bacteria susceptibility to antibiotics rely on a change in occupied area. Thus, a positive change in area indicates that the bacteria is growing and is resistant to the drug, right? Not always: Bacteria can also take on different shapes, such as filaments, or swell; these changes increase the area, but the bug is still susceptible to the antibiotic. Choi et al. therefore devised an imaging-based antibiotic susceptibility test (AST) that factors in changes in morphology, to rapidly determine whether single bacteria, confined to microwells, respond to various drugs. The authors looked at four standard strains, including antibiotic-resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA), as well as clinical samples, in response to dozens of different antibiotics used in the clinic. The single-cell morphology AST performed as well as the gold standard, culture-based broth microdilution test, with read-out in only 4 hours. Such a rapid and accurate screen could improve time-to-answer in the clinic by avoiding conventional culture methods, thus allowing for faster decision making in administering proper antibiotics to patients. A rapid antibiotic susceptibility test (AST) is desperately needed in clinical settings for fast and appropriate antibiotic administration. Traditional ASTs, which rely on cell culture, are not suitable for urgent cases of bacterial infection and antibiotic resistance owing to their relatively long test times. We describe a novel AST called single-cell morphological analysis (SCMA) that can determine antimicrobial susceptibility by automatically analyzing and categorizing morphological changes in single bacterial cells under various antimicrobial conditions. The SCMA was tested with four Clinical and Laboratory Standards Institute standard bacterial strains and 189 clinical samples, including extended-spectrum β-lactamase–positive Escherichia coli and Klebsiella pneumoniae, imipenem-resistant Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococci from hospitals. The results were compared with the gold standard broth microdilution test. The SCMA results were obtained in less than 4 hours, with 91.5% categorical agreement and 6.51% minor, 2.56% major, and 1.49% very major discrepancies. Thus, SCMA provides rapid and accurate antimicrobial susceptibility data that satisfy the recommended performance of the U.S. Food and Drug Administration.

Electrospray ionization-tandem mass spectrometry analysis of the mycolic acid profiles for the identification of common clinical isolates of mycobacterial species.

Mycolic acids are unique and complex molecular structures found in mycobacterial species. In the present study, we investigated whether electrospray ionization-tandem mass spectrometry (ESI-MS/MS) can be used to identify mycobacterial species based on their mycolic acid profiles. Clinical isolates of Mycobacterium tuberculosis complex and 18 nontuberculosis mycobacterial (NTM) species identified by PCR-restriction fragment length polymorphism (RFLP) or real-time PCR were used for this analysis. Crude lipid extracts were prepared by saponifying 1-2 colonies of individual isolates of mycobacterial species and by chloroform and methanol (2:1, v/v) extraction. ESI-MS/MS in negative ion mode with high cone voltage and collision energy was used for mycolic acid profiling analysis. Combinatorial precursor ion scans of m/z 395, 367, and 339 in the range of m/z 1000-1400 resulted in spectra specific to individual mycobacteria. M. tuberculosis complex and M. pulveris showed major ions by performing precursor ion scans on m/z 395 and 367, while other NTM species showed major ions by performing scans on m/z 367 and 339. The different NTM species examined showed different species dependent mycolic acid profiles. In conclusion, we describe a rapid, reliable, and informative ESI-MS/MS protocol for mycolic acid profiling in mycobacterial species, which allows mycobacterial species to be easily identified in clinical laboratories.

Evaluation of three phenotypic identification systems for clinical isolates of Raoultella ornithinolytica

Raoultella spp. have recently been separated from the genus Klebsiella based on their molecular characteristics. It was discovered that Raoultella ornithinolytica can be misidentified as Klebsiella oxytoca by commonly used phenotypic identification systems. Therefore, this study evaluated the ability of three phenotypic systems to identify R. ornithinolytica compared with the genotypic methods sequence-specific primer PCR (SSP-PCR), 16S rRNA gene sequence analysis using the MicroSeq 500 system16S rDNA bacterial identification system or comparison with GenBank sequences using blast. The phenotypic systems examined in this study were the VITEK 2 GN ID card, the MicroScan Neg Combo 32 panel and API 20E. The SSP-PCR panel was able to distinguish the R. ornithinolytica reference strain from other Raoultella spp. and K. oxytoca. Of the 27 isolates identified as R. ornithinolytica by SSP-PCR, VITEK 2 identified all of them as R. ornithinolytica. MicroScan and API identified 25 isolates (92.6%) and 24 isolates (88.9%) as K. oxytoca, respectively. These isolates were ornithine decarboxylase (ODC) negative in all three phenotypic systems. MicroSeq 500 identified 24 isolates (88.9%) as R. ornithinolytica, whereas GenBank identification was heterogeneous. Of the 68 isolates identified as K. oxytoca by SSP-PCR, 66 isolates (97.1%) were identified as K. oxytoca by VITEK 2, MicroScan and API. MicroScan and API require additional biochemical tests to differentiate between ODC-negative R. ornithinolytica and K. oxytoca.

Multiplex enzyme assay for galactosemia using ultraperformance liquid chromatography-tandem mass spectrometry.

BACKGROUND Galactosemia is one of the most important inherited disorders detected by newborn screening tests. Abnormal results in screening tests should be confirmed by enzyme activity assays, but existing methods are time and labor intensive. We developed a novel multiplex enzyme assay for galactosemia using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). METHODS [(13)C6]-galactose, [(13)C2]-galactose-1-phosphate, and UDP-glucose were used as substrates for 3 galactose-metabolizing enzymes. The end products from the combined reaction mixtures, [(13)C6]-galactose-1-phosphate, UDP-[(13)C2]-galactose, and UDP-galactose, were simultaneously measured using UPLC-MS/MS. Linearity, imprecision, ion suppression, and the effects of substrate were evaluated to determine assay performance. Enzyme activities from 35 healthy individuals, 8 patients with enzyme deficiency, and 18 mutant cells were analyzed. RESULTS Substrates, products, and internal standards from the mixture of 3 enzyme reactions were clearly separated by using UPLC-MS/MS, with an injection cycle time of 10 min. Ion suppression was 0.1%-2.5%, the interassay imprecision of UPLC-MS/MS was 3.3%-10.6% CV, and the linearity of each system was good (R(2) = 0.994-0.999). Patient samples and mutated cells showed consistently low enzyme activities compared with those of normal individuals and wild-type cells. CONCLUSIONS This method allows for a high-throughput and reproducible multiplex enzyme assay for galactosemia in erythrocytes.

Empirical Use of Ciprofloxacin for Acute Uncomplicated Pyelonephritis Caused by Escherichia coli in Communities Where the Prevalence of Fluoroquinolone Resistance Is High

ABSTRACT There is little information about the effectiveness of ciprofloxacin in regions where ciprofloxacin-resistant Escherichia coli is prevalent. This study was conducted to evaluate whether ciprofloxacin is effective as the initial empirical antibiotic for treatment of uncomplicated acute pyelonephritis (APN) due to ciprofloxacin-resistant E. coli. A total of 255 women with clinical diagnoses of uncomplicated APN due to E. coli were enrolled in the emergency department between March 2005 and December 2008. All enrolled patients were initially treated with ciprofloxacin. Patients were followed up 4 to 7 days after the start of therapy and 14 to 21 days after its completion. At the first follow-up visit, ciprofloxacin was changed to the appropriate antibiotic when necessary, depending on the antibiotic susceptibility results. Not only improvement of symptoms and signs but also microbiologic eradication was assessed at each visit. Fifteen percent (39/255) of the E. coli isolates were resistant to ciprofloxacin. There was no statistically significant difference between the clinical cure rates of the ciprofloxacin-susceptible group and the ciprofloxacin-resistant group at the first follow-up (87.0% versus 76.9%, P = 0.135) or the second follow-up (98.6% versus 94.9%, P = 0.177). However, there was a lower microbiologic cure rate in the ciprofloxacin-resistant group than in the ciprofloxacin-susceptible group (92.4% versus 41.7%, P = 0.000) at the first follow-up visit. No complications occurred in the ciprofloxacin-resistant group during the follow-up period. Our findings indicate that ciprofloxacin is an appropriate choice for empirical therapy of uncomplicated APN and has no serious adverse outcomes, if it is tailored appropriately, even for women infected with ciprofloxacin-resistant E. coli.

Human bloodstream infection caused by Staphylococcus pettenkoferi

Staphylococcus pettenkoferi is a recently isolated human pathogen with only a few reported cases of infection. We report a case of bloodstream infection caused by S. pettenkoferi in a patient with pulmonary tuberculosis.

Method for simultaneous analysis of nine second-line anti-tuberculosis drugs using UPLC-MS/MS

OBJECTIVES Therapeutic drug monitoring (TDM) of anti-tuberculosis (TB) drugs is beneficial for patients responding slowly to treatment and those with multidrug-resistant TB. We used ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to develop a rapid method for simultaneously measuring the blood concentrations of nine second-line anti-TB drugs: streptomycin, kanamycin, clarithromycin, cycloserine, moxifloxacin, levofloxacin, para-aminosalicylic acid, prothionamide and linezolid. METHODS Serum samples were extracted with acidified methanol and neutralized with NaOH. A Waters Acquity HSS T3 column and gradients of ammonium formate and acetonitrile in 0.1% formic acid were used for UPLC separation. Drug concentrations were determined by multiple reaction monitoring in positive ion mode, and assay performance was evaluated. We applied this method to TDM, analysing random serum samples from 85 patients treated with second-line drugs. RESULTS Sample preparation using acidified methanol extraction followed by neutralization yielded good recovery and ionization efficiency, with chromatographic separation achieved within 3 min per sample. Within-run and between-run precisions were 1.7%-7.5% and 1.7%-12.4%, respectively, at concentrations representing low and high levels for the nine drugs. Lower limits of detection and quantification were 0.025-0.5 and 0.25-5.0 μg/mL, respectively. Linearity was acceptable at five concentrations for each drug. No ion suppression was observed at the retention time for most compounds, except for streptomycin, kanamycin and cycloserine, which were eluted close to the void volume of the column. In a limited pilot study, all quantifiable human samples had values within the validated assay ranges. CONCLUSIONS The performance of our MS/MS detection technique was generally acceptable. The method provided rapid, sensitive and reproducible quantification of nine second-line anti-TB drugs and should facilitate drug monitoring during treatment.

Ultra‐performance liquid chromatography/tandem mass spectrometry for determination of sulfatides in dried blood spots from patients with metachromatic leukodystrophy

RATIONALE Metachromatic leukodystrophy (MLD) is a genetic autosomal recessive disease caused by a deficiency in arylsulfatase A. Accumulated sulfatides can be detected in the urine and detection of sulfatiduria is a useful test for diagnosis and monitoring. To our knowledge, no studies have explored the accumulation of sulfatides in dried blood spots (DBSs). We developed an ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method for measuring sulfatides in DBSs from patients with MLD. METHODS DBSs were eluted with internal standard. After mixing and centrifugation, the organic layer was transferred to a 96-well microplate and dried, then resuspended in methanol/propanol solution. Samples were analyzed on an UPLC system. Total running time was 4 min. Quantification was achieved by multiple reaction monitoring using a tandem mass spectrometer. We evaluated the precision, linearity, and ion suppression of the method and analyzed sulfatide concentrations in DBS specimens from MLD patients (n = 9), pseudodeficiency (PD) patient (n = 1), obligate heterozygotes (OH) (n = 2) and normal controls (n = 124). RESULTS In negative-ion mode, sulfatides species subjected to collision-induced dissociation readily fragment to produce an intense ion at m/z 96.8 (HSO4(-)). The precisions of low and high concentration controls ranged from 5.4 to 19.9%. The sulfatides produced linear responses. Molecular species of sulfatides were barely detected in DBSs from normal individuals and the PD-OH group [mean (range), 0.07 (<0.05-0.34) and 0.13 (<0.05-0.22) µg/mL, respectively]. In contrast, the DBSs from MLD patients showed a marked increase in several molecular species of sulfatide [mean (range), 2.02 (1.18-3.89) µg/mL]. CONCLUSIONS Simultaneous detection for sulfatides using UPLC/MS/MS can be successfully applied to DBS analysis. This method provides a fast and effective screening and monitoring tool for the diagnosis and treatment of MLD.

[Detection of mycobacterium tuberculosis complex using real-time polymerase chain reaction].

BACKGROUND For the detection of Mycobacterium tuberculosis complex (MTB), PCR is known to be sensitive, specific, and rapid compared to the conventional methods of acid-fast-bacilli (AFB) smear and culture. We evaluated a new approach for MTB detection using real-time PCR. METHODS The specificity of real-time PCR was evaluated using 20 MTB isolates and 37 nontuberculous mycobacteria (NTM) isolates identified by AccuProbe Mycobacterium tuberculosis complex colony identification test (Gen-Probe Inc., USA) and Myco-ID (M&D, Korea). One hundred sputum specimens (50 AFB smear-positive and 50 negative specimens) were analyzed using real-time PCR and Amplicor Mycobacterium tuberculosis test (Roche, Germany). The results of real-time PCR positives (55 samples) and negatives (598 samples) were analyzed by AFB smear and culture. RESULTS The real-time PCR assay accurately discriminated between MTB and NTM species. Realtime PCR and Amplicor test yielded the same results in 96.0% (96/100) of the sputum specimens tested. The sensitivity and specificity of real-time PCR based on AFB culture were 97.4% and 88.5%, respectively. Of the 55 real-time PCR positive specimens, 83.6% (46/55) were culture-positive, 30.9% (17/55) were smear-positive, 52.7% (29/55) were smear-negative and culture-positive, and 14.5% (8/55) were both smear and culture-negative. Among the 598 real-time PCR negative specimens, 60 were not tested for AFB smear or culture and 10 were contaminated. Of the remaining 528 specimens, 478 (90.5%) were both smear and culture-negative and 39 (7.4%) were culture-positive. CONCLUSIONS For the detection of MTB, real-time PCR was sensitive and specific and comparable to conventional methods. It can be used for rapid identification of M. tuberculosis in clinical laboratories.

CD36 polymorphism and its relationship with body mass index and coronary artery disease in a Korean population.

Abstract Background: CD36 is a multifunctional membrane receptor and a cell-adhesion molecule that is expressed in platelets, monocytes/macrophages, microvascular endothelial cells, cardiac monocytes and adipocytes. In this study, we investigated whether genetic polymorphisms of the CD36 gene are associated with risk of coronary artery disease (CAD) in a Korean population. Methods: PCR and polyacrylamide gel electrophoresis or PCR-restriction fragment length polymorphism assays were performed to analyze the following CD36 gene polymorphisms: a (TG) repeat in intron 3 and the base substitution 478C>T (Pro90Ser). A total of 219 patients with significant CAD and 236 control subjects were examined with regard to their genotypes, lipid profiles and other risk factors for CAD. Results: The frequency of (TG) 11- or 12-repeat homozygotes was significantly higher in male CAD patients than in control men (28.4% vs. 15.7%, OR=2.13, p=0.018). Homozygosity for the (TG) 11- or 12-repeat allele was also significantly associated with a higher body mass index (BMI) compared to non-carriers in 134 control men after controlling for age, smoking and hypertension, and explains a 13% BMI variation observed in this study (p=0.015, analysis of covariance). For the 478C>T mutation, which has been reported to be associated with CD36 deficiency, there was no difference in the frequency of the 478T allele between CAD patients and control subjects. However, the 478T allele was found to be closely linked with a (TG) 11- or 12-repeat allele of intron 3 in the control subjects (χ2=18.88, p<0.001). Conclusions: The (TG) repeat polymorphism in intron 3 of the CD36 gene is associated with a higher BMI and cardiovascular risk for men in a Korean population. Clin Chem Lab Med 2007;45:1277–82.