Rapid pathogen-specific phenotypic antibiotic susceptibility testing using digital LAMP quantification in clinical samples

Rapid antimicrobial susceptibility testing (AST) is urgently needed for informing treatment decisions and preventing the spread of antimicrobial resistance resulting from the misuse and overuse of antibiotics. To date, no phenotypic AST exists that can be performed within a single patient visit (30 min) directly from clinical samples. We show that AST results can be obtained by using digital nucleic acid quantification to measure the phenotypic response of Escherichia coli present within clinical urine samples exposed to an antibiotic for 15 min. We performed this rapid AST using our ultrafast (~7 min) digital real-time loop-mediated isothermal amplification (dLAMP) assay [area under the curve (AUC), 0.96] and compared the results to a commercial (~2 hours) digital polymerase chain reaction assay (AUC, 0.98). The rapid dLAMP assay can be used with SlipChip microfluidic devices to determine the phenotypic antibiotic susceptibility of E. coli directly from clinical urine samples in less than 30 min. With further development for additional pathogens, antibiotics, and sample types, rapid digital AST (dAST) could enable rapid clinical decision-making, improve management of infectious diseases, and facilitate antimicrobial stewardship

Digital Quantification of DNA Replication and Chromosome Segregation Enables Determination of Antimicrobial Susceptibility after only 15 Minutes of Antibiotic Exposure

Rapid antimicrobial susceptibility testing (AST) would decrease misuse and overuse of antibiotics. The “holy grail” of AST is a phenotype-based test that can be performed within a doctor visit. Such a test requires the ability to determine a pathogen's susceptibility after only a short antibiotic exposure. Herein, digital PCR (dPCR) was employed to test whether measuring DNA replication of the target pathogen through digital single-molecule counting would shorten the required time of antibiotic exposure. Partitioning bacterial chromosomal DNA into many small volumes during dPCR enabled AST results after short exposure times by 1) precise quantification and 2) a measurement of how antibiotics affect the states of macromolecular assembly of bacterial chromosomes. This digital AST (dAST) determined susceptibility of clinical isolates from urinary tract infections (UTIs) after 15 min of exposure for all four antibiotic classes relevant to UTIs. This work lays the foundation to develop a rapid, point-of-care AST and strengthen global antibiotic stewardship

Rapid pathogen-specific phenotypic antibiotic susceptibility testing using digital LAMP quantification in clinical samples

Rapid antimicrobial susceptibility testing (AST) is urgently needed for informing treatment decisions and preventing the spread of antimicrobial resistance resulting from the misuse and overuse of antibiotics. To date, no phenotypic AST exists that can be performed within a single patient visit (30 min) directly from clinical samples. We show that AST results can be obtained by using digital nucleic acid quantification to measure the phenotypic response of Escherichia coli present within clinical urine samples exposed to an antibiotic for 15 min. We performed this rapid AST using our ultrafast (~7 min) digital real-time loop-mediated isothermal amplification (dLAMP) assay [area under the curve (AUC), 0.96] and compared the results to a commercial (~2 hours) digital polymerase chain reaction assay (AUC, 0.98). The rapid dLAMP assay can be used with SlipChip microfluidic devices to determine the phenotypic antibiotic susceptibility of E. coli directly from clinical urine samples in less than 30 min. With further development for additional pathogens, antibiotics, and sample types, rapid digital AST (dAST) could enable rapid clinical decision-making, improve management of infectious diseases, and facilitate antimicrobial stewardship

Temporal analysis of natural variation for the rate of leaf production and its relationship with flowering initiation in Arabidopsis thaliana

Vegetative growth and flowering initiation are two crucial developmental processes in the life cycle of annual plants that are closely associated. The timing of both processes affects several presumed adaptive traits, such as flowering time (FT), total leaf number (TLN), or the rate of leaf production (RLP). However, the interactions among these complex processes and traits, and their mechanistic bases, remain largely unknown. To determine the genetic relationships between them, the natural genetic variation between A. thaliana accessions Fei-0 and Ler has been studied using a new population of 222 Ler×Fei-0 recombinant inbred lines. Temporal analysis of the parental development under a short day photoperiod distinguishes two vegetative phases differing in their RLP. QTL mapping of RLP in consecutive time intervals of vegetative development indicates that Ler/Fei-0 variation is caused by 10 loci whose small to moderate effects mainly display two different temporal patterns. Further comparative QTL analyses show that most of the genomic regions affecting FT or TLN also alter RLP. In addition, the partially independent genetic bases observed for FT and TLN appear determined by several genomic regions with two different patterns of phenotypic effects: regions with a larger effect on FT than TLN, and vice versa. The distinct temporal and pleiotropic patterns of QTL effects suggest that natural variation for flowering time is caused by different genetic mechanisms involved in vegetative and/or reproductive phase changes, most of them interacting with the control of leaf production rate. Thus, natural selection might contribute to maintain this genetic variation due to its phenotypic effects not only on the timing of flowering initiation but also on the rate of vegetative growth

Major-Effect Alleles at Relatively Few Loci Underlie Distinct Vernalization and Flowering Variation in Arabidopsis Accessions

We have explored the genetic basis of variation in vernalization requirement and response in Arabidopsis accessions, selected on the basis of their phenotypic distinctiveness. Phenotyping of F2 populations in different environments, plus fine mapping, indicated possible causative genes. Our data support the identification of FRI and FLC as candidates for the major-effect QTL underlying variation in vernalization response, and identify a weak FLC allele, caused by a Mutator-like transposon, contributing to flowering time variation in two N. American accessions. They also reveal a number of additional QTL that contribute to flowering time variation after saturating vernalization. One of these was the result of expression variation at the FT locus. Overall, our data suggest that distinct phenotypic variation in the vernalization and flowering response of Arabidopsis accessions is accounted for by variation that has arisen independently at relatively few major-effect loci

Tnpa Trans-Activates Methylated Maize Suppressor-Mutator Transposable Elements in Transgenic Tobacco

The maize Suppressor-mutator (Spm) transposable element is subject to epigenetic inactivation in transgenic tobacco, as it is in maize. Spm inactivation in tobacco is correlated with increased methylation of sequences near the element's transcription start site. To determine whether element-encoded gene products can promote the reactivation of an inactive element, we investigated the effects of introducing individual CaMV 35S promoter-driven cDNAs for tnpA, tnpB, tnpC and tnpD, the element's four known protein-coding sequences. Introduction of the tnpA cDNA promoted the reactivation of the inactive resident Spm element, as judged by the appearance of regenerants with very early excision events and transposed elements. By contrast, the tnpB, tnpC and tnpD cDNAs had no affect on the activity of the resident Spm element. Similar results were obtained when the element-encoded cDNAs were introduced either by Agrobacterium-mediated retransformation or by a genetic cross. Reactivation of an inactive Spm by the tnpA cDNA is accompanied by reduced methylation of several methylation-sensitive restriction sites near the element's transcription start site. Maintenance of the reactivated Spm element in an active state requires the continued presence of the tnpA cDNA. Elimination of the tnpA cDNA locus by genetic segregation generally results in decreased element activity, as judged by a low frequency of excision events, and is accompanied by increased methylation of the element's 5'-end. Exceptions resembling the phenomenon of ``presetting'' are also observed in which progeny plants that did not receive the tnpA cDNA locus after meiotic segregation maintain high excision activity and exhibit low methylation levels