A fatal outbreak of neonatal sepsis caused by mcr-10-carrying Enterobacter kobei in a tertiary care hospital in Nepal.

BACKGROUND: Enterobacter kobei is an emerging cause of outbreak of nosocomial infections in neonatal intensive care units (NICUs). Between July and September 2016, a NICU in a tertiary care hospital of Nepal observed an abrupt increase in the number of neonatal sepsis cases caused by Enterobacter spp. infecting 11 out of 23 admitted neonates, five of whom died of an exacerbated sepsis. AIM: To confirm the suspected outbreak, identify environmental source of infection, and characterize genetic determinants of antimicrobial resistance (AMR) and virulence of the pathogen. METHODS: Whole-genome sequencing of all Enterobacter spp. isolated from blood cultures of septic neonates admitted to NICU between May 2016 and December 2017 was performed. Also, an environmental sampling was intensified from fortnightly to weekly during the outbreak. FINDINGS: The genomic analysis revealed that 10 out of 11 non-duplicated E. kobei isolated from neonatal blood cultures between July and September 2016 were clonal, confirming the outbreak. The isolates carried AMR genes including blaAmpC and mcr-10 conferring reduced susceptibility to carbapenem and colistin respectively. The environmental sampling, however, failed to isolate any Enterobacter spp. Reinforcement of aseptic protocols in invasive procedures, hand hygiene, environmental decontamination, fumigation, and secluded care of culture-positive cases successfully terminated the outbreak. CONCLUSION: Our study underscored the need to implement stringent infection control measures to prevent infection outbreaks. For the first time, we report the emergence of carbapenem and colistin non-susceptible E. kobei carrying mcr-10 gene as a cause of nosocomial neonatal sepsis in a NICU.

An Improved Calibration Approach for Traveling Wave Ion Mobility Spectrometry: Robust, High-Precision Collision Cross Sections.

The combination of ion-mobility (IM) separation with mass spectrometry (MS) has impacted global measurement efforts in areas ranging from food analysis to drug discovery. Reasons for the broad adoption of IM-MS include its significantly increased peak capacity, duty-cycle, and ability to reconstruct fragmentation data in parallel, all of which greatly enable the analyses of complex mixtures. More fundamentally, however, measurements of ion-gas molecule collision cross sections (CCSs) are used to support compound identification and quantitation efforts as well as study the structures of large biomolecules. As the first commercialized form of IM-MS, Traveling Wave Ion Mobility (TWIM) devices are operated at low pressures (∼3 mbar) and voltages, are relatively short (∼25 cm), and separate ions on a timescale of tens of milliseconds. These qualities make TWIM ideally suited for hybridization with MS. Owing to the complicated motion of ions in TWIM devices, however, IM transit times must be calibrated to enable CCS measurements. Applicability of these calibrations has hitherto been restricted to primarily singly charged small molecules and some classes of large, multiply charged ions under a significantly narrower range of instrument conditions. Here, we introduce and extensively characterize a dramatically improved TWIM calibration methodology. Using over 2500 experimental TWIM data sets, covering ions that span over 3.5 orders of magnitude of molecular mass, we demonstrate robust calibrations for a significantly expanded range of instrument conditions, thereby opening up new analytical application areas and enabling the expansion of high-precision CCS measurements for both existing and next-generation TWIM instrumentation.

Bifunctional cross-linking approaches for mass spectrometry-based investigation of nucleic acids and protein-nucleic acid assemblies.

With the goal of expanding the very limited toolkit of cross-linking agents available for nucleic acids and their protein complexes, we evaluated the merits of a wide range of bifunctional agents that may be capable of reacting with the functional groups characteristic of these types of biopolymers. The survey specifically focused on the ability of test reagents to produce desirable inter-molecular conjugates, which could reveal the identity of interacting components and the position of mutual contacts, while also considering a series of practical criteria for their utilization as viable nucleic acid probes. The survey employed models consisting of DNA, RNA, and corresponding protein complexes to mimic as close as possible typical applications. Denaturing polyacrylamide gel electrophoresis (PAGE) and mass spectrometric (MS) analyses were implemented in concert to monitor the formation of the desired conjugates. In particular, the former was used as a rapid and inexpensive tool for the efficient evaluation of cross-linker activity under a broad range of experimental conditions. The latter was applied after preliminary rounds of reaction optimization to enable full-fledged product characterization and, more significantly, differentiation between mono-functional and intra- versus inter-molecular conjugates. This information provided the feedback necessary to further optimize reaction conditions and explain possible outcomes. Among the reagents tested in the study, platinum complexes and nitrogen mustards manifested the most favorable characteristics for practical cross-linking applications, whereas other compounds provided inferior yields, or produced rather unstable conjugates that did not survive the selected analytical conditions. The observed outcomes will help guide the selection of the most appropriate cross-linking reagent for a specific task, whereas the experimental conditions described here will provide an excellent starting point for approaching these types of applications. As a whole, the results of the survey clearly emphasize that finding a universal reagent, which may afford excellent performance with all types of nucleic acid substrates, will require extending the exploration beyond the traditional chemistries employed to modify the constitutive functional groups of these vital biopolymers.