Biology subjects

Engelthaler, D. M.

Publications and source records attributed to Engelthaler, D. M..

7 recordsLinked to original sources

bioRxiv2025

Unpacking bedaquiline hetero-resistance: the importance of intermediate profiles for phenotypic drug-susceptibility testing

Phenotypic drug susceptibility testing (pDST) remains the gold standard for Mycobacterium tuberculosis complex drug resistance determination. Next generation sequencing technologies can identify heteroresistant populations at low frequencies, but little is known about the impact of heteroresistance on bedaquiline (BDQ) pDST results. We simulated heteroresistance using in vitro generated MmpR5 mutants mixed with the progenitor strain at various percentages (1-20%) and did pDST using MGIT960 culture (1 and 2 {micro}g/mL BDQ concentrations). Targeted Next Generation Sequencing (tNGS) was used to quantify the mutant sub-population in growth control tubes, which were expected to maintain the mutant: wild type proportion throughout the assay. Growth units of these growth control tubes were also comparable with minor differences in time-to-positivity between ratio mixtures. Only when intermediate results were considered could BDQ heteroresistance be detected at frequencies of approximately 1% by pDST at a critical concentration of 1 {micro}g/mL using BACTEC MGIT960 coupled with EpiCenter TBeXiST software. The ability of pDST, a widely available DST technique, to reveal the presence of BDQ-resistant subpopulations at the phenotypic testing stage could improve resistance determination and potentially reduce time to effective treatment. ImportanceMultidrug resistant tuberculosis (MDR-TB) is estimated to cause up to 19% of all antimicrobial resistance-attributable deaths worldwide. Further, the success rate for the treatment of drug-resistant TB, in the presence of adherence, is poor at only 68%. The advent of bedaquiline (BDQ) has revolutionized MDR-TB care, but BDQ resistance determination is hampered by several obstacles facing both phenotypic and genotypic testing. Specifically for phenotypic susceptibility testing, BDQ-resistant Mycobacterium tuberculosis isolates with variants in MmpR5, which may display minimal inhibitory concentration values just below the critical concentration or are present at low frequencies (heteroresistance; the presence of mixed mutant and wild-type populations within a specimen), are typically designated as susceptible. This may lead to prescription of an ineffective regimen and amplification of resistance. The BACTEC MGIT960 platform coupled with EpiCenter TBeXiST software for phenotypic testing, which is currently the only routinely used method of BDQ DST, can be used to derive more information about underlying resistant populations. We demonstrate how this is possible through the consideration of intermediate results (i.e., when growth units in a drug-containing tube reach the threshold for resistance but only after a further week of incubation). These intermediate results, commonly disregarded by TB laboratories, could lead to earlier detection of BDQ resistance. This is especially crucial when the genetic mechanism of resistance is unknown, a variant has not been associated with resistance in the interim, and in cases of heteroresistance.

microbiology↗

bioRxiv2024

Discovery and Repurposing of Pharmaceutical Agents as Antifungals: In vivo activity against Coccidioides

There is significant interest in identifying improved treatments for coccidioidomycosis, an endemic fungal infection found in the southwestern United States, and Central and South America. The current standard of care, fluconazole, often fails to completely eradicate Coccidioides infection; however, the cost of identifying new compounds is often high in terms of both finances and time. Therefore, repurposing existing pharmaceutical agents is an attractive option. In our previous work, we identified several compounds which inhibited fungal growth in vitro. Based on these findings, we screened a subset of these agents to increase the performance of fluconazole in a combination therapy approach, as compared to fluconazole alone, in a murine model. We observed that combination therapy of sertraline:fluconazole significantly reduced the amount of live fungus in the lung compared to fluconazole alone. These results suggest that sertraline can be repurposed as an adjunctive agent in the treatment of this important fungal disease.

microbiology↗

bioRxiv2023

Lyssa excreta: Defining parameters for fecal samples as a rabies virus surveillance method

It is not possible to systematically screen the environment for rabies virus (RABV) using current approaches. We sought to determine under what conditions RABV is detectable from feces and other accessible samples from infected wildlife to broaden the number of biological samples that could be used to test for RABV. We employed a recently-developed quantitative RT-PCR assay called the "LN34 panlyssavirus real-time RT-PCR assay", which is highly sensitive and specific for all variants of RABV. We harvested and tested brain tissue, fecal, and/or mouth swab samples from 25 confirmed RABV positive bats of six species. To determine if rabies RNA lasts in feces sufficiently long post-defecation to use it as a surveillance tool, we tested fecal samples from 10 bats at the time of sample collection and after 24 hours of exposure to ambient conditions, with an additional test on six bats out to 72 hours. To assess whether we could pool fecal pellets and still detect a positive, we generated dilutions of known positives at 1:1, 1:10, 1:50, and 1:200. For six individuals for which matched brain, mouth swab, and fecal samples were tested, results were positive for 100%, 67%, and 67%, respectively. For the first time test to 24 hours, 63% of feces that were positive at time 0 were still positive after 24 hours, and 50% of samples at 72 hours were positive across all three replicates. Pooling tests revealed that fecal positives were detected at 1:10 dilution, but not at 1:50 or 1:200. Our preliminary results suggest that fecal samples hold promise for a rapid and non-invasive environmental screening system.

pathology↗

bioRxiv2020

Phylogenetic Analysis of St. Louis Encephalitis Virus within Two Southwestern United State Counties: a case for a bulk introduction event into the southwest United States

St. Louis Encephalitis Virus (SLEV) has been seasonally detected within the Culex spp. populations within Maricopa County, Arizona and Coachella Valley, California since an outbreak in Maricopa County in 2015. Previous work revealed that the outbreak was caused by an importation of SLEV genotype III, which had only been detected within Argentina in prior years. However, little is known about when the importation occurred or the population dynamics since its arrival into the southwestern United States. In this study, we wanted to determine if the annual detection of SLEV in Maricopa and Riverside counties is due to enzootic cycling or new importations. To address this question, we analyzed 143 SLEV genomes (138 sequenced as part of this study) using the Bayesian phylogenetic analysis software, BEAST, to estimate the date of arrival into the American Southwest and characterize the underlying population structure of SLEV. Phylogenetic clustering showed that SLEV variants circulating in Arizona and California form two distinct populations with little evidence of transmission among the two populations since the onset of the outbreak. Interestingly, the SLEV variants in Coachella Valley appear to be annually imported from a nearby source, whereas the Arizona population is locally sourced each year. Finally, the earliest representatives of SLEV genotype III in the southwestern US formed a polytomy that includes both California and Arizona samples. We propose that the initial outbreak could have resulted from an introductory population of SLEV, perhaps in one or more bird flocks migrating north in 2013, rather than a single variant introduced by one bird.

bioinformatics↗

bioRxiv2019

Bacterial Genome wide association studies (bGWAS) and transcriptomics identifies cryptic antimicrobial resistance mechanisms in Acinetobacter baumannii

Antimicrobial resistance (AMR) in the nosocomial pathogen, Acinetobacter baumannii, is becoming a serious public health threat. While some mechanisms of AMR have been reported, understanding novel mechanisms of resistance is critical for identifying emerging resistance. One of the first steps in identifying novel AMR mechanisms is performing genotype/phenotype association studies. However, performing genotype/phenotype association studies is complicated by the plastic nature of the A. baumannii pan-genome. In this study, we compared the antibiograms of 12 antimicrobials associated with multiple drug families for 84 A. baumannii isolates, many isolated in Arizona, USA. in silico screening of these genomes for known AMR mechanisms failed to identify clear correlations for most drugs. We then performed a genome wide association study (GWAS) looking for associations between all possible 21-mers; this approach generally failed to identify mechanisms that explained the resistance phenotype. In order to decrease the genomic noise associated with population stratification, we compared four phylogenetically-related pairs of isolates with differing susceptibility profiles. RNA-Sequencing (RNA-Seq) was performed on paired isolates and differentially expressed genes were identified. In these isolate pairs, we identified four different potential mechanisms, highlighting the difficulty of broad AMR surveillance in this species. To verify and validate differential expression, amplicon sequencing was performed. These results suggest that a diagnostic platform based on gene expression rather than genomics alone may be beneficial in certain surveillance efforts. The implementation of such advanced diagnostics coupled with increased AMR surveillance will potentially improve A. baumannii infection treatment and patient outcomes.

genomics↗

bioRxiv2019

Genomic variant identification methods alter Mycobacterium tuberculosis transmission inference

Pathogen genomic data are increasingly used to characterize global and local transmission patterns of important human pathogens and to inform public health interventions. Yet there is no current consensus on how to measure genomic variation. We investigated the effects of variant identification approaches on transmission inferences for M. tuberculosis by comparing variants identified by five different groups in the same sequence data from a clonal outbreak. We then measured the performance of commonly used variant calling approaches in recovering variation in a simulated tuberculosis outbreak and tested the effect of applying increasingly stringent filters on transmission inferences and phylogenies. We found that variant calling approaches used by different groups do not recover consistent sets of variants, often leading to conflicting transmission inferences. Further, performance in recovering true outbreak variation varied widely across approaches. Finally, stringent filters rapidly eroded the accuracy of transmission inferences and quality of phylogenies reconstructed from outbreak variation. We conclude that measurements of genetic distance and phylogenetic structure are dependent on variant calling approach. Variant calling algorithms trained upon true sequence data outperform other approaches and enable inclusion of repetitive regions typically excluded from genomic epidemiology studies, maximizing the information gleaned from outbreak genomes.

genomics↗

bioRxiv2017

Phylogenetic analysis of West Nile Virus in Maricopa County, Arizona: Evidence for dynamic behavior of strains in two major lineages in the American Southwest

West Nile Virus (WNV) has been detected annually in Maricopa County, Arizona, since 2003. With this in mind, we sought to determine if contemporary strains are established within the county or are annually imported. As part of this effort, we developed a new protocol for tiled amplicon sequencing of WNV to efficiently attain greater than 99% coverage of 14 WNV genomes collected directly from positive mosquito pools distributed throughout Maricopa County between 2014 and 2017. Bayesian phylogenetic analyses revealed that the contemporary genomes fall within two major lineages, NA/WN02 and SW/WN03. We found that all of the Arizona strains possessed a mutation known to be under positive selection (NS5-K314R), which has arisen independently four times. The SW/WN03 strains exhibited transient behavior, with at least 10 separate introductions into Arizona when considering both historical and contemporary strains. However, NA/WN02 strains are geographically differentiated and appear to be established in Arizona, with likely origins in New York. The clade of New York and Arizona strains looks to be the most ancestral extant lineage of WNV still circulating in the United States. The establishment of WNV strains in Maricopa County provides the first evidence of local overwintering by a WNV strain over the course of several years in Arizona.

evolutionary biology↗