Biology subjects

Kreiswirth, B. N.

Publications and source records attributed to Kreiswirth, B. N..

9 recordsLinked to original sources

bioRxiv2025

The Quantification of Drug Accumulation within Gram-Negative Bacteria

Intrabacterial drug accumulation, mediated by the bacterial permeability barrier, efflux, and intrabacterial drug metabolism, is of general significance to the interaction between small molecules and bacteria. For example, the ability of a small molecule to accumulate within a bacterium influences its ability to serve as a chemical probe of an intracellular protein target and/or its efficacy as an antibacterial drug discovery entity. A general method to quantitatively interrogate both intrabacterial drug accumulation and metabolism (IBDM) is presented for Gram-negative bacteria and exemplified with Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa in both single-compound and high-throughput formats. The liquid chromatography-mass spectrometry based platform does not depend on drug labelling and its utility is highlighted through the demonstrated correlation of drug accumulation with drug minimum inhibitory concentration (MIC) in both wild type and efflux deficient strains of E. coli and a matched pair of K. pneumoniae clinical and laboratory strains of varying degrees of drug resistance. Furthermore, an investigation of drug synergy implicates the selective enhancement of the accumulation of one drug by its partner therapy. Finally, a high-throughput format is validated and deployed which provides a readily adaptable approach to screening assays. We anticipate the further applications of this platform to both the translational and the fundamental studies of the interactions of small molecules with bacteria.

microbiology↗

bioRxiv2024

In vitro and in vivo validation of cwlM and pbpB essentiality for viability and resistance to imipenem in Mycobacterium abscessus

Mycobacterium abscessus lung infection is notoriously difficult to treat due, in part, to the intrinsic resistance of this pathogen to most marketed antibiotics. {beta}-Lactams, namely imipenem and cefoxitin, are first-line drugs in combination regimens used to treat this infection; and there is growing interest in dual-{beta}-lactam-based regimens. Better understanding of the molecular basis of {beta}-lactam activity through study of the genetic determinants of {beta}-lactam susceptibility and tolerance would enable more rational drug combinations and guide discovery of novel drug targets to complement {beta}-lactams. We recently used an inducible CRISPR interference (CRISPRi) system to silence cwlM and pbpB and confirm their essentiality for in vitro growth and resistance to sub-MIC concentrations of imipenem. Here, we extend those findings to show that silencing either gene alone is bactericidal and augments the bactericidal activity of imipenem in vitro. Furthermore, using CRISPRi in a mouse model of M. abscessus lung infection for the first time, we confirm the essentiality of each gene for in vivo survival. These results validate cwlM and pbpB as essential genes and promising drug targets in this pathogen, including for potentiation of carbapenem activity. The results further establish CRISPRi as a powerful method for validating drug targets and studying gene-gene and gene-drug interactions in vitro and in vivo.

microbiology↗

bioRxiv2022

Pre-epidemic evolution of the USA300 clade and a molecular key for classification

USA300 has remained the dominant community and healthcare associated methicillin-resistant Staphylococcus aureus (MRSA) clone in the United States and in northern South America for at least the past 20 years. In this time, it has experienced epidemic spread in both of these locations. However, its pre-epidemic evolutionary history and origins are incompletely understood. Large sequencing databases, such as NCBI, PATRIC, and Staphopia, contain clues to the early evolution of USA300 in the form of sequenced genomes of USA300 isolates that are representative of lineages that diverged prior to the establishment of the South American (SAE) and North American (NAE) epidemics. In addition, historical isolates collected prior to the emergence of epidemics can help reconstruct early events in the history of this lineage. Here, we take advantage of the accrued, publicly available data, as well as two newly sequenced pre-epidemic historical isolates from 1996, and a very early diverging ACME-negative NAE genome to understand the pre-epidemic evolution of USA300. We use database mining techniques to emphasize genomes similar to pre-epidemic isolates, with the goal of reconstructing the early molecular evolution of the USA300 lineage. Phylogenetic analysis with these genomes confirms that the North American Epidemic and South American Epidemic USA300 lineages diverged from a most recent common ancestor around 1970 with high confidence, and it also pinpoints the independent acquisition events of the of the ACME and COMER loci with greater precision than in previous studies. We solidify evidence for a North American origin of the USA300 lineage and identify multiple introductions of USA300 into South America from North America. Notably, we describe a third major USA300 clade (the pre-epidemic branching clade; PEB1) consisting of both MSSA and MRSA isolates circulating around the world that diverged from the USA300 lineage prior to the establishment of the South American and North American epidemics. We present a detailed analysis of specific sequence characteristics of each of the major clades, and present diagnostic positions that can be used to classify new genomes.

genomics↗

bioRxiv2022

Within-host genotypic and phenotypic diversity of contemporaneous carbapenem-resistant Klebsiella pneumoniae from blood cultures of patients with bacteremia

Carbapenem-resistant Klebsiella pneumoniae (CRKP) are major pathogens globally. It is unknown whether bloodstream infections (BSIs) by CRKP and other bacteria are commonly caused by single organisms or mixed microbial populations. We hypothesized that contemporaneous CRKP from blood cultures of individual patients are genetically and phenotypically distinct. We determined short-read whole genome sequences of 10 strains from single colonies from CRKP-positive blood cultures in each of 6 patients (Illumina HiSeq). All strains were sequence type (ST)-258 K. pneumoniae that were unique by core genome single nucleotide polymorphism phylogeny, antibiotic resistance and virulence genes, capsular polysaccharide (CPS) gene mutations, and/or plasmid loss. Strains from each of 3 patients that differed in antibiotic resistance, virulence and/or CPS gene content underwent long-read sequencing for genome completion (Oxford Nanopore), and were tested for phenotypes in vitro and pathogenicity during mouse BSIs. Genetically distinct strains within individual patients exhibited significant differences in carbapenem, beta-lactam/beta-lactamase inhibitor and other antibiotic responses, CPS production, mucoviscosity, and susceptibility to serum killing. In 2 patients, strains differed significantly in their ability to infect organs and cause mortality in mice. In conclusion, we identified genotypic and phenotypic variant ST258 K. pneumoniae strains from blood cultures of individual patients, which were not detected by the clinical laboratory at time of BSI diagnosis. The data support a new paradigm of CRKP population diversity during BSIs. If validated for other BSIs, within-host bacterial diversity may have profound implications for medical, microbiology laboratory and infection prevention practices, and for understanding emergence of antibiotic resistance and pathogenesis. IMPORTANCEIn processing positive microbiologic cultures, standard clinical laboratory practice is to test a single bacterial strain from each morphologically distinct colony. We performed comprehensive whole genome sequence analyses on 10 carbapenem-resistant Klebsiella pneumoniae (CRKP) strains from positive blood cultures from each of 6 patients. Our findings that all strains were genetically unique and that genetic variants manifested differences in phenotypes like antibiotic responsiveness and virulence suggest that CRKP bloodstream infections may be commonly caused by mixed bacterial populations. Results raise questions about laboratory protocols and treatment decisions that are directed against a single strain. The observation that pan-genome analyses revealed inter-strain differences that were not evident by studying core genomes has important implications for investigating nosocomial outbreaks and transmission. Data also suggest a model of pathogenesis of CRKP infections, in which environmental pressures in vivo may select for outgrowth of variants that manifest antibiotic resistance, tolerance or specific virulence attributes.

microbiology↗

bioRxiv2021

What do we gain when tolerating loss? The information bottleneck, lossy compression, and detecting horizontal gene transfer

Most microbes have the capacity to acquire genetic material from their environment. Recombination of foreign DNA yields genomes that are, at least in part, incongruent with the vertical history of their species. Dominant approaches for detecting these transfers are phylogenetic, requiring a painstaking series of analyses including alignment and phylogenetic tree reconstruction. These traditional pan-genomic methods do not scale. Here we propose an unsupervised, alignment-free and tree-free technique based on the sequential information bottleneck (SIB), an optimization procedure designed to extract some portion of relevant information from one random variable conditioned on another. In our case, this joint probability distribution tabulates occurrence counts of k-mers against their genomes of origin with the expectation that recombination will create a strong signal that unifies certain sets of co-occuring k-mers. We conceptualize the technique as a rate-distortion problem, measuring distortion in the relevance information as k-mers are compressed into clusters based on their co-occurrence in the source genomes. The result is fast, model-free, lossy compression of k-mers into groups that learns tracts of shared genome sequence differentiating recombined elements from the vertically inherited core. We show that the technique yields a new recombination measure based purely on information, divorced from any biases and limitations inherent to alignment and phylogeny. SignificanceThe information bottleneck, a lossy compression technique borrowed from the information theoretic and Natural Langauge Processing literature, is well suited to detecting evolutionary patterns in sets of co-occuring k-mers. Here we show that we can detect simulated and real recombination events while highlighting a core set of k-mers that comprise the vertically inherited portion of any set of genomes. Moreover, the compressibility of any given set of genomes offers a new way to compare the pangenomes of clades across the microbial tree of life. In our application, the bottleneck is informed by genome origin, our relevance variable, but the technique is general. The information bottleneck can be used for any biological contingency matrix where the goal is to learn groups from unstructured data.

evolutionary biology↗

bioRxiv2020

Genetic composition and evolution of the prevalent Mycobacterium tuberculosis lineages 2 and 4 in the Chinese and Zhejiang Province populations

The causative agent of tuberculosis (TB) comprises seven human-adapted lineages. Human movements and host genetics are crucial to TB dissemination. We analyzed whole-genome sequencing data for a countrywide collection of 1154 isolates and a provincial collection of 1296 isolates, constructed the best-scoring maximum likelihood phylogenetic tree, conducted Bayesian evolutionary analysis to compute the most recent common ancestors of lineages 2 and 4, and assessed the antigenic diversity in human T cell epitopes by calculating pairwise dN/dS ratios. Of the 1296 Zhejiang isolates, 964 (74.38%) belonged to lineage 2 and 332 (25.62%) belonged to lineage 4. L2.2 is the most ancient sub-lineage in Zhejiang, first appearing approximately 6897 years ago (95% HDI: 6513-7298). L4.4 is the most modern sub-lineage, first appearing approximately 2217 years ago (95% HDI: 1864-2581). The dN/dS ratios revealed that the epitope and non-epitope regions of lineage 2 strains were significantly (P<0.001) more conserved than those of lineage 4. An increase in the frequency of lineage 4 may reflect its successful transmission over the last 20 years. The recent common ancestors and transmission routes of the sub-lineages are related to the entry of humans into China and Zhejiang Province.

molecular biology↗

bioRxiv2018

In vitro activity of the new β-lactamase inhibitors relebactam and vaborbactam in combination with β-lactams against Mycobacterium abscessus complex clinical isolates

Pulmonary disease due to infection with Mycobacterium abscessus complex (MABC) is notoriously difficult to treat, in large part due to MABCs intrinsic resistance to most antibiotics, including {beta}-lactams. MABC organisms express a broad-spectrum {beta}-lactamase that is resistant to traditional {beta}-lactam-based {beta}-lactamase inhibitors but inhibited by a newer non-{beta}-lactam-based {beta}-lactamase inhibitor, avibactam. Consequently, the susceptibility of MABC to some {beta}-lactams is increased in the presence of avibactam. Therefore, we hypothesized that two new non-{beta}-lactam-based {beta}-lactamase inhibitors, relebactam and vaborbactam, would also increase susceptibility of MABC to {beta}-lactams. The objective of the present study was to evaluate the in vitro activity of various marketed {beta}-lactams alone and in combination with either relebactam or vaborbactam against multidrug-resistant MABC clinical isolates. Our data demonstrate that both {beta}-lactamase inhibitors significantly improved the anti-MABC activity of many carbapenems (including imipenem and meropenem) and cephalosporins (including cefepime, ceftaroline, and cefuroxime). As a meropenem/vaborbactam combination is now marketed and an imipenem/relebactam combination is currently in phase III trials, these fixed combinations may become the {beta}-lactams of choice for the treatment of MABC infections. Furthermore, given the evolving interest in dual {beta}-lactam regimens, our results identify select cephalosporins, such as cefuroxime, with superior activity in the presence of a {beta}-lactamase inhibitor, deserving of further evaluation in combination with these carbapenem/{beta}-lactamase inhibitor products.

microbiology↗

bioRxiv2018

Molecular epidemiology of carbapenem-resistant Enterobacter cloacae complex infections uncovers high frequency of non-carbapenemase-producers in five tertiary care hospitals from Colombia

BackgroundInfections caused by carbapenem-resistant Enterobacter cloacae (CR-Ecl) have been increasingly reported in the clinical setting; here we describe the clinical and molecular characteristics of CR-Ecl infections in a KPC endemic region.\n\nMethodsA cross-sectional study was conducted in five tertiary-care hospitals in Medellin-Colombia. All patients infected by CR-Ecl from June-2012 to June-2014 were included. Sociodemographics and clinical information was retrieved from medical records. Antimicrobial susceptibility testing, phenotypic and molecular carbapenemase detection were performed. Analysis of hsp60 and PFGE was done in a subset of isolates.\n\nResultsOf 109 patients enrolled, 60.55% (66/109) were infected with non-carbapenemase-producing-Ecl (non-CP-Ecl). CP-Ecl patients were frequently hospitalized in the ICU (37.21% vs 12.12%) and had exposure to carbapenems (39.53% vs 15.15%) compared to non-CP-Ecl infected patients. All-cause 30-day mortality was higher in CP-Ecl than non-CP-Ecl infected patients (27.91% vs 19.70%). CP-Ecl harbored KPC-2 (83.72%) and KPC-3 (6.97%). Analysis of hsp60 showed that CP-Ecl belonged primarily to cluster-VI of Enterobacter xiangfangensis (12/34) and cluster-XI (12/34) corresponding to E. cloacae subsp. cloacae. Non-CP-Ecl isolates belonged to cluster-VII/VIII (45/54), of E. hormaechi subsp. steigerwaltii. PFGE revealed isolates in cluster VII/VIII and XI were closely related within their own clusters.\n\nConclusionsThe results revealed a high frequency of non-CP-Ecl among the CR-Ecl infections in a KPC endemic region, displaying distinct clinical and molecular characteristics in comparison to CP-Ecl. The study highlights a significant contribution of non-CP-Ecl to the prevalence of CR-Ecl. Infection control measures to curtail dissemination of CR-Ecl should not only focus on CP-Ecl but should also include non-CP-Ecl.

epidemiology↗

bioRxiv2018

Enterobacter cloacae complex ST171 Isolates Expressing KPC-4 Carbapenemase Recovered from Canine Patients in Ohio, USA

Carbapenem resistant Enterobacteriaceae (CRE) have emerged as a critical public health threat. Organisms expressing the Klebsiella pneumoniae carbapenemase (KPC) were first recognized in the US in the late 1990s and continue to be the predominant CRE genotype reported in clinical isolates. Strains harboring blaKPC alleles have been observed in multiple species of Enterobacteriaceae, including the Enterobacter cloacae complex. A major E. cloacae clone, Enterobacter xiangfangensis ST171, has emerged as an important cause of hospital associated infections (HAI) and has been shown to carry different alleles of KPC in the context of Tn4401, residing on plasmids of multiple incompatibility groups. While CRE are commonly isolated from infected humans, their recovery from animals has been rare, particularly from companion animals. In the US, only six CRE have been reported from companion animals, and one from livestock, none of which were blaKPC. This report describes two E. xiangfangensis sequence type ST171 isolates each with a large IncHI2 plasmid bearing blaKPC-4 recovered from dogs with infections at the Ohio State University Veterinary Medical Center. Our phylogenetic comparison of these canine isolates with available sequences from clinical human isolates of KPC-4 identified in ST171 suggest an epidemiologically significant clonal strain.

epidemiology↗