Bacterial droplet-based single-cell RNA-seq reveals antibiotic-associated heterogeneous cellular states.

We introduce BacDrop, a highly scalable technology for bacterial single-cell RNA sequencing that has overcome many challenges hindering the development of scRNA-seq in bacteria. BacDrop can be applied to thousands to millions of cells from both gram-negative and gram-positive species. It features universal ribosomal RNA depletion and combinatorial barcodes that enable multiplexing and massively parallel sequencing. We applied BacDrop to study Klebsiella pneumoniae clinical isolates and to elucidate their heterogeneous responses to antibiotic stress. In an unperturbed population presumed to be homogeneous, we found within-population heterogeneity largely driven by the expression of mobile genetic elements that promote the evolution of antibiotic resistance. Under antibiotic perturbation, BacDrop revealed transcriptionally distinct subpopulations associated with different phenotypic outcomes including antibiotic persistence. BacDrop thus can capture cellular states that cannot be detected by bulk RNA-seq, which will unlock new microbiological insights into bacterial responses to perturbations and larger bacterial communities such as the microbiome.

Inter-species geographic signatures for tracing horizontal gene transfer and long-term persistence of carbapenem resistance.

BACKGROUND: Carbapenem-resistant Enterobacterales (CRE) are an urgent global health threat. Inferring the dynamics of local CRE dissemination is currently limited by our inability to confidently trace the spread of resistance determinants to unrelated bacterial hosts. Whole-genome sequence comparison is useful for identifying CRE clonal transmission and outbreaks, but high-frequency horizontal gene transfer (HGT) of carbapenem resistance genes and subsequent genome rearrangement complicate tracing the local persistence and mobilization of these genes across organisms. METHODS: To overcome this limitation, we developed a new approach to identify recent HGT of large, near-identical plasmid segments across species boundaries, which also allowed us to overcome technical challenges with genome assembly. We applied this to complete and near-complete genome assemblies to examine the local spread of CRE in a systematic, prospective collection of all CRE, as well as time- and species-matched carbapenem-susceptible Enterobacterales, isolated from patients from four US hospitals over nearly 5 years. RESULTS: Our CRE collection comprised a diverse range of species, lineages, and carbapenem resistance mechanisms, many of which were encoded on a variety of promiscuous plasmid types. We found and quantified rearrangement, persistence, and repeated transfer of plasmid segments, including those harboring carbapenemases, between organisms over multiple years. Some plasmid segments were found to be strongly associated with specific locales, thus representing geographic signatures that make it possible to trace recent and localized HGT events. Functional analysis of these signatures revealed genes commonly found in plasmids of nosocomial pathogens, such as functions required for plasmid retention and spread, as well survival against a variety of antibiotic and antiseptics common to the hospital environment. CONCLUSIONS: Collectively, the framework we developed provides a clearer, high-resolution picture of the epidemiology of antibiotic resistance importation, spread, and persistence in patients and healthcare networks.

Genetic determinants facilitating the evolution of resistance to carbapenem antibiotics.

In this era of rising antibiotic resistance, in contrast to our increasing understanding of mechanisms that cause resistance, our understanding of mechanisms that influence the propensity to evolve resistance remains limited. Here, we identified genetic factors that facilitate the evolution of resistance to carbapenems, the antibiotic of 'last resort', in Klebsiella pneumoniae, the major carbapenem-resistant species. In clinical isolates, we found that high-level transposon insertional mutagenesis plays an important role in contributing to high-level resistance frequencies in several major and emerging carbapenem-resistant lineages. A broader spectrum of resistance-conferring mutations for select carbapenems such as ertapenem also enables higher resistance frequencies and, importantly, creates stepping-stones to achieve high-level resistance to all carbapenems. These mutational mechanisms can contribute to the evolution of resistance, in conjunction with the loss of systems that restrict horizontal resistance gene uptake, such as the CRISPR-Cas system. Given the need for greater antibiotic stewardship, these findings argue that in addition to considering the current efficacy of an antibiotic for a clinical isolate in antibiotic selection, considerations of future efficacy are also important. The genetic background of a clinical isolate and the exact antibiotic identity can and should also be considered as they are determinants of a strain's propensity to become resistant. Together, these findings thus provide a molecular framework for understanding acquisition of carbapenem resistance in K. pneumoniae with important implications for diagnosing and treating this important class of pathogens.

Mechanism of threshold size assessment: Metamorphosis is triggered by the TGF-beta/Activin ligand Myoglianin.

Although the mechanisms that control growth are now well understood, the mechanism by which animals assess their body size remains one of the great puzzles in biology. The final larval instar of holometabolous insects, after which growth stops and metamorphosis begins, is specified by a threshold size. We investigated the mechanism of threshold size assessment in the tobacco hornworm, Manduca sexta. The threshold size was found to change depending on the amount of exposure to poor nutrient conditions whereas hypoxia treatment consistently led to a lower threshold size. Under these various conditions, the mass of the muscles plus integuments was correlated with the threshold size. Furthermore, the expression of myoglianin (myo) increased at the threshold size in both M. sexta and Tribolium castaneum. Knockdown of myo in T. castaneum led to larvae that underwent supernumerary larval molts and stayed in the larval stage permanently even after passing the threshold size. We propose that increasing levels of Myo produced by the growing tissues allow larvae to assess their body size and trigger metamorphosis at the threshold size.

Simultaneous detection of genotype and phenotype enables rapid and accurate antibiotic susceptibility determination.

Multidrug resistant organisms are a serious threat to human health1,2. Fast, accurate antibiotic susceptibility testing (AST) is a critical need in addressing escalating antibiotic resistance, since delays in identifying multidrug resistant organisms increase mortality3,4 and use of broad-spectrum antibiotics, further selecting for resistant organisms. Yet current growth-based AST assays, such as broth microdilution5, require several days before informing key clinical decisions. Rapid AST would transform the care of patients with infection while ensuring that our antibiotic arsenal is deployed as efficiently as possible. Growth-based assays are fundamentally constrained in speed by doubling time of the pathogen, and genotypic assays are limited by the ever-growing diversity and complexity of bacterial antibiotic resistance mechanisms. Here we describe a rapid assay for combined genotypic and phenotypic AST through RNA detection, GoPhAST-R, that classifies strains with 94-99% accuracy by coupling machine learning analysis of early antibiotic-induced transcriptional changes with simultaneous detection of key genetic resistance determinants to increase accuracy of resistance detection, facilitate molecular epidemiology and enable early detection of emerging resistance mechanisms. This two-pronged approach provides phenotypic AST 24-36 h faster than standard workflows, with <4 h assay time on a pilot instrument for hybridization-based multiplexed RNA detection implemented directly from positive blood cultures.