Rapid Characterization of Bacterial Lipids with Ambient Ionization Mass Spectrometry for Species Differentiation.

Ambient ionization mass spectrometry (AIMS) is both labor and time saving and has been proven to be useful for the rapid delineation of trace organic and biological compounds with minimal sample pretreatment. Herein, an analytical platform of probe sampling combined with a thermal desorption-electrospray ionization/mass spectrometry (TD-ESI/MS) and multivariate statistical analysis was developed to rapidly differentiate bacterial species based on the differences in their lipid profiles. For comparison, protein fingerprinting was also performed with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) to distinguish these bacterial species. Ten bacterial species, including five Gram-negative and five Gram-positive bacteria, were cultured, and the lipids in the colonies were characterized with TD-ESI/MS. As sample pretreatment was unnecessary, the analysis of the lipids in a bacterial colony growing on a Petri dish was completed within 1 min. The TD-ESI/MS results were further performed by principal component analysis (PCA) and hierarchical cluster analysis (HCA) to assist the classification of the bacteria, and a low relative standard deviation (5.2%) of the total ion current was obtained from repeated analyses of the lipids in a single bacterial colony. The PCA and HCA results indicated that different bacterial species were successfully distinguished by the differences in their lipid profiles as validated by the differences in their protein profiles recorded from the MALDI-TOF analysis. In addition, real-time monitoring of the changes in the specific lipids of a colony with growth time was also achieved with probe sampling and TD-ESI/MS. The developed analytical platform is promising as a useful diagnostic tool by which to rapidly distinguish bacterial species in clinical practice.

Emergence of a predominant sequence type ST7363 and the increasing trend of resistance to cefixime and ceftriaxone in Neisseria gonorrhoeae in Southern Taiwan, 2019-2021.

BACKGROUND/PURPOSE: Multi-drug resistance and the presence of epidemic lineages of Neisseria gonorrhoeae locally and globally were important clinical and public health issues. We aimed to investigate the molecular epidemiology and the antimicrobial susceptibility profiles of N. gonorrhoeae in Southern Taiwan. METHODS: Between 2019 and 2021, adult patients who had suspected gonorrhea and attended a urology clinic in southern Taiwan were recruited to participate in this study. Clinical data from medical records and a questionnaire, antimicrobial susceptibility testing using a disk diffusion test in accordance with the guidelines by the Clinical and Laboratory Standards Institute, and Multi-locus sequence typing (MLST) were analyzed. RESULTS: A total of 500 patients participated in the surveillance study. Among them, 232 N. gonorrhoeae isolates were identified, but only 164 isolates were recovered for further research. ST7363 (n = 83, 50.61%) was found to be the predominant sequence type, followed by ST1583 (n = 24, 14.63%), ST1588 (n = 13, 7.93%), and ST7827 (n = 12, 7.32%). 100% resistance to penicillin and 99.4% non-susceptible rate of ciprofloxacin were observed. The azithromycin resistant rate being 15.24% and the cefixime non-susceptible rate being 17.07% were alarming, both with decreasing trends in susceptibilities during 2019-2021. The 25 azithromycin resistant isolates were mainly belonged to ST7363 (n = 12) and ST7827 (n = 3). Seven (4.2%) isolates were ceftriaxone non-susceptible. Among them, four were assigned to be ST 7827 and three belonged to ST7363. CONCLUSION: We observed the emergence of a predominant sequence type ST7363 in southern Taiwan. Compared with previous Taiwan studies, the increasing trend of resistance to cefixime and ceftriaxone necessitates clinicians' alertness for clinical treatment response of the extended spectrum cephalosporins and the further surveillance monitor.

A novel, rapid (within hours) culture-free diagnostic method for detecting live Mycobacterium tuberculosis with high sensitivity.

BACKGROUND: Nucleic acid amplification tests (NAATs) are widely used to diagnose tuberculosis (TB), but cannot discriminate live bacilli from dead bacilli. Live bacilli can be isolated by culture methods, but this is time-consuming. We developed a de novo TB diagnostic method that detects only live bacilli with high sensitivity within hours. METHODS: A prospective study was performed in Taiwan from 2017 to 2018. Sputum was collected consecutively from 1102 patients with suspected TB infection. The sputum was pretreated and heated at 46°C for 1 h to induce the secretion of MPT64 protein from live Mycobacterium tuberculosis. MPT64 was detected with our ultrasensitive enzyme-linked immunosorbent assay (ELISA) coupled with thionicotinamide-adenine dinucleotide (thio-NAD) cycling. We compared our data with those obtained using a culture test (MGIT), a smear test (Kinyoun staining), and a NAAT (Xpert). FINDINGS: The limit of detection for MPT64 in our culture-free ultrasensitive ELISA was 2.0 × 10-19 moles/assay. When the criterion for a positive response was set as an absorbance value ≥17 mAbs, this value corresponded to ca. 330 CFU/mL in the culture method - almost the same high-detection sensitivity as the culture method. To confirm that MPT64 is secreted from only live bacilli, M. bovis BCG was killed using 8 µg/mL rifampicin and then heated. Following this procedure, our method detected no MPT64. Our rapid ultra-sensitive ELISA-based method required only 5 h to complete. Comparing the results of our method with those of culture tests for 944 specimens revealed a sensitivity of 86.9% (93/107, 95% CI: 79.0-92.7%) and a specificity of 92.0% (770/837, 95% CI: 89.9-93.7%). The performance data were not significantly different (McNemar's test, P = 0.887) from those of the Xpert tests. In addition, at a ≥1+ titer in the smear test, the positive predictive value of our culture-free ultrasensitive ELISA tests was in a good agreement with that of the culture tests. Furthermore, our culture-free ultrasensitive ELISA test had better validity for drug effectiveness examination than Xpert tests because our test detected only live bacilli. INTERPRETATION: Our culture-free ultrasensitive ELISA method detects only live TB bacilli with high sensitivity within hours, allowing for rapid diagnosis of TB and monitoring drug efficacy. FUNDING: Matching Planner Program from JST (VP29117939087), the A-STEP Program from JST (AS3015096U), Waseda University grants for Specific Research Projects (2017A-015 and 2019C-123), the Precise Measurement Technology Promotion Foundation to E.I.