Identifying Clinically Relevant Bacteria Directly from Culture and Clinical Samples with a Handheld Mass Spectrometry Probe.

BACKGROUND: Rapid identification of bacteria is critical to prevent antimicrobial resistance and ensure positive patient outcomes. We have developed the MasSpec Pen, a handheld mass spectrometry-based device that enables rapid analysis of biological samples. Here, we evaluated the MasSpec Pen for identification of bacteria from culture and clinical samples. METHODS: A total of 247 molecular profiles were obtained from 43 well-characterized strains of 8 bacteria species that are clinically relevant to osteoarticular infections, including Staphylococcus aureus, Group A and B Streptococcus, and Kingella kingae, using the MasSpec Pen coupled to a high-resolution mass spectrometer. The molecular profiles were used to generate statistical classifiers based on metabolites that were predictive of Gram stain category, genus, and species. Then, we directly analyzed samples from 4 patients, including surgical specimens and clinical isolates, and used the classifiers to predict the etiologic agent. RESULTS: High accuracies were achieved for all levels of classification with a mean accuracy of 93.3% considering training and validation sets. Several biomolecules were detected at varied abundances between classes, many of which were selected as predictive features in the classifiers including glycerophospholipids and quorum-sensing molecules. The classifiers also enabled correct identification of Gram stain type and genus of the etiologic agent from 3 surgical specimens and all classification levels for clinical specimen isolates. CONCLUSIONS: The MasSpec Pen enables identification of several bacteria at different taxonomic levels in seconds from cultured samples and has potential for culture-independent identification of bacteria directly from clinical samples based on the detection of metabolic species.

Study on the Gas-Phase Reactivity of Charged Pyridynes.

The reactivities of three isomeric, charged ortho-pyridynes, the 1,2-, 2,3-, and 3,4-didehydropyridinium cations, were examined in the gas phase using Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometry. The structures of selected product ions were probed using collision-activated dissociation (CAD) experiments in a linear quadrupole ion trap (LQIT) mass spectrometer. Mechanisms based on quantum chemical calculations are proposed for the formation of all major products. The products of the reactions of the charged ortho-pyridynes in the gas phase were found to closely resemble those formed upon reactions of neutral ortho-arynes in solution, but the mechanisms of these reactions exhibit striking differences. Additionally, no radical reactions were observed for any of the charged ortho-pyridynes examined, in contrast to previous proposals that ortho-benzyne can occasionally react via radical mechanisms. Finally, the relative reactivities of those charged gaseous ortho-pyridynes that yielded similar product distributions were found to be affected mainly by the (calculated) vertical electron affinities of the dehydrocarbon sites, which suggests that the reactivity of these species is controlled by polar effects.

Simultaneous quantitation of five triazole anti-fungal agents by paper spray-mass spectrometry.

Background Invasive fungal disease is a life-threatening condition that can be challenging to treat due to pathogen resistance, drug toxicity, and therapeutic failure secondary to suboptimal drug concentrations. Frequent therapeutic drug monitoring (TDM) is required for some anti-fungal agents to overcome these issues. Unfortunately, TDM at the institutional level is difficult, and samples are often sent to a commercial reference laboratory for analysis. To address this gap, the first paper spray-mass spectrometry assay for the simultaneous quantitation of five triazoles was developed. Methods Calibration curves for fluconazole, posaconazole, itraconazole, hydroxyitraconazole, and voriconazole were created utilizing plasma-based calibrants and four stable isotopic internal standards. No sample preparation was needed. Plasma samples were spotted on a paper substrate in pre-manufactured plastic cartridges, and the dried plasma spots were analyzed directly utilizing paper spray-mass spectrometry (paper spray MS/MS). All experiments were performed on a Thermo Scientific TSQ Vantage triple quadrupole mass spectrometer. Results The calibration curves for the five anti-fungal agents showed good linearity (R2 = 0.98-1.00). The measured assay ranges (lower limit of quantification [LLOQ]-upper limit of quantitation [ULOQ]) for fluconazole, posaconazole, itraconazole, hydroxyitraconazole, and voriconazole were 0.5-50 µg/mL, 0.1-10 µg/mL, 0.1-10 µg/mL, 0.1-10 µg/mL, and 0.1-10 µg/mL, respectively. The inter- and intra-day accuracy and precision were less than 25% over the respective ranges. Conclusions We developed the first rapid paper spray-MS/MS assay for simultaneous quantitation of five triazole anti-fungal agents in plasma. The method may be a powerful tool for near-point-of-care TDM aimed at improving patient care by reducing the turnaround time and for use in clinical research.

16S rRNA deep sequencing identifies Actinotignum schaalii as the major component of a polymicrobial intra-abdominal infection and implicates a urinary source.

Introduction. It can be difficult to catalogue the individual organisms comprising polymicrobial patient infections, both because conventional clinical microbiological culture does not facilitate the isolation and enumeration of all members of a complex microbial community, and because fastidious organisms may be mixed with organisms that grow rapidly in vitro. Empiric antimicrobial treatment is frequently employed based on the anatomical site and the suspected source of the infection, especially when an appropriately collected surgical specimen is not obtained. Case presentation. We present a case of an intra-abdominal infection in a patient with complex anatomy and recurrent urinary tract infections. Imaging did not reveal a clear source of infection, no growth was obtained from urine cultures and initial abdominal fluid cultures were also negative. In contrast, 16S rRNA deep sequencing of abdominal fluid samples revealed mixed bacterial populations with abundant anaerobes, including Actinotignum schaalii (Actinobaculum schaalii). Ultimately, only Enterobacter cloacae complex and meticillin-resistant Staphylococcus aureus, both of which were identified by sequencing, were recovered by culture. Conclusion. The clinical application of 16S rRNA deep sequencing can more comprehensively and accurately define the organisms present in an individual patient's polymicrobial infection than conventional microbiological culture, detecting species that are not recovered under standard culture conditions or that are otherwise unexpected. These results can facilitate effective antimicrobial stewardship and help elucidate the possible origins of infections.

Experimental and computational studies on the formation of three para-benzyne analogues in the gas phase.

Experimental and computational studies on the formation of three gaseous, positively-charged para-benzyne analogues in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer are reported. The structures of the cations were examined by isolating them and allowing them to react with various neutral reagents whose reactions with aromatic carbon-centered σ-type mono- and biradicals are well understood. Cleavage of two iodine-carbon bonds in N-deuterated 1,4-diiodoisoquinolinium cation by collision-activated dissociation (CAD) produced a long-lived cation that showed nonradical reactivity, which was unexpected for a para-benzyne. However, the reactivity closely resembles that of an isomeric enediyne, N-deuterated 2-ethynylbenzonitrilium cation. A theoretical study on possible rearrangement reactions occurring during CAD revealed that the cation formed upon the first iodine atom loss undergoes ring-opening before the second iodine atom loss to form an enediyne instead of a para-benzyne. Similar results were obtained for the 5,8-didehydroisoquinolinium cation and the 2,5-didehydropyridinium cation. The findings for the 5,8-didehydroisoquinolinium cation are in contradiction with an earlier report on this cation. The cation described in the literature was regenerated by using the literature method and demonstrated to be the isomeric 5,7-didehydro-isoquinolinium cation and not the expected 5,8-isomer.

Does the 2,6-didehydropyridinium cation exist?

Reactive intermediates are key species involved in many chemical and biochemical processes. For example, carbon-centered aromatic σ,σ-biradicals formed in biological systems from naturally occurring enediyne antitumor antibiotics are responsible for the irreversible cleavage of double-stranded DNA caused by these prodrugs. However, because of their high reactivity, it is very difficult or impossible to isolate and investigate these biradicals. The aromatic σ,σ-biradical, 2,6-didehydropyridine, has been speculated for many years to be formed in certain organic reactions; however, no definitive proof of its generation has been obtained. We report here the successful generation of protonated 2,6-didehydropyridine and the examination of its chemical properties in the gas phase by using a Fourier transform ion cyclotron resonance mass spectrometer. The results suggest that a mixture of singlet (ground) state and triplet (excited) state 2,6-didehydropyridinium cations was generated. The two different states show qualitatively different reactivity, with the triplet state showing greater Brønsted acidity than that of the singlet state. The triplet state also shows much greater radical reactivity than that of the singlet state, as expected because of the coupling of the nonbonding electrons in the singlet state.