TheiaEuk: a species-agnostic bioinformatics workflow for fungal genomic characterization.

Introduction: The clinical incidence of antimicrobial-resistant fungal infections has dramatically increased in recent years. Certain fungal pathogens colonize various body cavities, leading to life-threatening bloodstream infections. However, the identification and characterization of fungal isolates in laboratories remain a significant diagnostic challenge in medicine and public health. Whole-genome sequencing provides an unbiased and uniform identification pipeline for fungal pathogens but most bioinformatic analysis pipelines focus on prokaryotic species. To this end, TheiaEuk_Illumina_PE_PHB (TheiaEuk) was designed to focus on genomic analysis specialized to fungal pathogens. Methods: TheiaEuk was designed using containerized components and written in the workflow description language (WDL) to facilitate deployment on the cloud-based open bioinformatics platform Terra. This species-agnostic workflow enables the analysis of fungal genomes without requiring coding, thereby reducing the entry barrier for laboratory scientists. To demonstrate the usefulness of this pipeline, an ongoing outbreak of C. auris in southern Nevada was investigated. We performed whole-genome sequence analysis of 752 new C. auris isolates from this outbreak. Furthermore, TheiaEuk was utilized to observe the accumulation of mutations in the FKS1 gene over the course of the outbreak, highlighting the utility of TheiaEuk as a monitor of emerging public health threats when combined with whole-genome sequencing surveillance of fungal pathogens. Results: A primary result of this work is a curated fungal database containing 5,667 unique genomes representing 245 species. TheiaEuk also incorporates taxon-specific submodules for specific species, including clade-typing for Candida auris (C. auris). In addition, for several fungal species, it performs dynamic reference genome selection and variant calling, reporting mutations found in genes currently associated with antifungal resistance (FKS1, ERG11, FUR1). Using genome assemblies from the ATCC Mycology collection, the taxonomic identification module used by TheiaEuk correctly assigned genomes to the species level in 126/135 (93.3%) instances and to the genus level in 131/135 (97%) of instances, and provided zero false calls. Application of TheiaEuk to actual specimens obtained in the course of work at a local public health laboratory resulted in 13/15 (86.7%) correct calls at the species level, with 2/15 called at the genus level. It made zero incorrect calls. TheiaEuk accurately assessed clade type of Candida auris in 297/302 (98.3%) of instances. Discussion: TheiaEuk demonstrated effectiveness in identifying fungal species from whole genome sequence. It further showed accuracy in both clade-typing of C. auris and in the identification of mutations known to associate with drug resistance in that organism.

The use of whole-genome sequencing and development of bioinformatics to monitor overlapping outbreaks of Candida auris in southern Nevada.

A Candida auris outbreak has been ongoing in Southern Nevada since August 2021. In this manuscript we describe the sequencing of over 200 C. auris isolates from patients at several facilities. Genetically distinct subgroups of C. auris were detected from Clade I (3 distinct lineages) and III (1 lineage). Open-source bioinformatic tools were developed and implemented to aid in the epidemiological investigation. The work herein compares three methods for C. auris whole genome analysis: Nullarbor, MycoSNP and a new pipeline TheiaEuk. We also describe a novel analysis method focused on elucidating phylogenetic linkages between isolates within an ongoing outbreak. Moreover, this study places the ongoing outbreaks in a global context utilizing existing sequences provided worldwide. Lastly, we describe how the generated results were communicated to the epidemiologists and infection control to generate public health interventions.

The role of plasmids in carbapenem resistant E. coli in Alameda County, California.

BACKGROUND: Antimicrobial resistant infections continue to be a leading global public health crisis. Mobile genetic elements, such as plasmids, have been shown to play a major role in the dissemination of antimicrobial resistance (AMR) genes. Despite its ongoing threat to human health, surveillance of AMR in the United States is often limited to phenotypic resistance. Genomic analyses are important to better understand the underlying resistance mechanisms, assess risk, and implement appropriate prevention strategies. This study aimed to investigate the extent of plasmid mediated antimicrobial resistance that can be inferred from short read sequences of carbapenem resistant E. coli (CR-Ec) in Alameda County, California. E. coli isolates from healthcare locations in Alameda County were sequenced using an Illumina MiSeq and assembled with Unicycler. Genomes were categorized according to predefined multilocus sequence typing (MLST) and core genome multilocus sequence typing (cgMLST) schemes. Resistance genes were identified and corresponding contigs were predicted to be plasmid-borne or chromosome-borne using two bioinformatic tools (MOB-suite and mlplasmids). RESULTS: Among 82 of CR-Ec identified between 2017 and 2019, twenty-five sequence types (STs) were detected. ST131 was the most prominent (n = 17) followed closely by ST405 (n = 12). blaCTX-M were the most common ESBL genes and just over half (18/30) of these genes were predicted to be plasmid-borne by both MOB-suite and mlplasmids. Three genetically related groups of E. coli isolates were identified with cgMLST. One of the groups contained an isolate with a chromosome-borne blaCTX-M-15 gene and an isolate with a plasmid-borne blaCTX-M-15 gene. CONCLUSIONS: This study provides insights into the dominant clonal groups driving carbapenem resistant E. coli infections in Alameda County, CA, USA clinical sites and highlights the relevance of whole-genome sequencing in routine local genomic surveillance. The finding of multi-drug resistant plasmids harboring high-risk resistance genes is of concern as it indicates a risk of dissemination to previously susceptible clonal groups, potentially complicating clinical and public health intervention.

Analysis of SARS-CoV-2 variants from patient specimens in Nevada from October 2020 to August 2021.

In early 2020, the emergence and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the human population quickly developed into a global pandemic. SARS-CoV-2 is the etiological agent of coronavirus disease 2019 (COVID-19) which has a broad range of respiratory illnesses. As the virus circulates, it acquires nucleotide changes. These mutations are potentially due to the inherent differences in the selection pressures within the human population compared to the original zoonotic reservoir of SARS-CoV-2 and formerly naïve humans. The acquired mutations will most likely be neutral, but some may have implications for viral transmission, disease severity, and resistance to therapies or vaccines. This is a follow-up study from our early report (Hartley et al. J Genet Genomics. 01202021;48(1):40-51) which detected a rare variant (nsp12, RdRp P323F) circulating within Nevada in mid 2020 at high frequency. The primary goals of the current study were to determine the phylogenetic relationship of the SARS-CoV-2 genomes within Nevada and to determine if there are any unusual variants within Nevada compared to the current database of SARS-CoV-2 sequences. Whole genome sequencing and analysis of SARS-CoV-2 from 425 positively identified nasopharyngeal/nasal swab specimens were performed from October 2020 to August 2021 to determine any variants that could result in potential escape from current therapeutics. Our analysis focused on nucleotide mutations that generated amino acid variations in the viral Spike (S) protein, Receptor binding domain (RBD), and the RNA-dependent RNA-polymerase (RdRp) complex. The data indicate that SARS-CoV-2 sequences from Nevada did not contain any unusual variants that had not been previously reported. Additionally, we did not detect the previously identified the RdRp P323F variant in any of the samples. This suggests that the rare variant we detected before was only able to circulate because of the stay-at-home orders and semi-isolation experience during the early months of the pandemic. IMPORTANCE: SARS-COV-2 continues to circulate in the human population. In this study, SARS-CoV-2 positive nasopharyngeal/nasal swab samples were used for whole genome sequencing to determine the phylogenetic relationship of SARS-CoV-2 sequences within Nevada from October 2020 to August 2021. The resulting data is being added to a continually growing database of SARS-CoV-2 sequences that will be important for understanding the transmission and evolution of the virus as it spreads around the globe.

GAMBIT (Genomic Approximation Method for Bacterial Identification and Tracking): A methodology to rapidly leverage whole genome sequencing of bacterial isolates for clinical identification.

Whole genome sequencing (WGS) of clinical bacterial isolates has the potential to transform the fields of diagnostics and public health. To realize this potential, bioinformatic software that reports identification results needs to be developed that meets the quality standards of a diagnostic test. We developed GAMBIT (Genomic Approximation Method for Bacterial Identification and Tracking) using k-mer based strategies for identification of bacteria based on WGS reads. GAMBIT incorporates this algorithm with a highly curated searchable database of 48,224 genomes. Herein, we describe validation of the scoring methodology, parameter robustness, establishment of confidence thresholds and the curation of the reference database. We assessed GAMBIT by way of validation studies when it was deployed as a laboratory-developed test in two public health laboratories. This method greatly reduces or eliminates false identifications which are often detrimental in a clinical setting.

Rapid Lineage Assignment of Severe Acute Respiratory Syndrome Coronavirus 2 Cases through Automated Library Preparation, Sequencing, and Bioinformatic Analysis.

The coronavirus disease 2019 (COVID-19) pandemic has provided a stage to illustrate that there is considerable value in obtaining rapid, whole-genome-based information about pathogens. This article describes the utility of a commercially available, automated severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) library preparation, genome sequencing, and a bioinformatics analysis pipeline to provide rapid, near-real-time SARS-CoV-2 variant description. This study evaluated the turnaround time, accuracy, and other quality-related parameters obtained from commercially available automated sequencing instrumentation, from analysis of continuous clinical samples obtained from January 1, 2021, to October 6, 2021. This analysis included a base-by-base assessment of sequencing accuracy at every position in the SARS-CoV-2 chromosome using two commercially available methods. Mean turnaround time, from the receipt of a specimen for SARS-CoV-2 testing to the availability of the results, with lineage assignment, was <3 days. Accuracy of sequencing by one method was 100%, although certain sites on the genome were found repeatedly to have been sequenced with varying degrees of read error rate.

Clonal Lineages and Virulence Factors of Carbapenem Resistant E. coli in Alameda County, California, 2017-2019.

The prevalence of carbapenem-resistant Enterobacterales (CRE) has been increasing since the year 2000 and is considered a serious public health threat according to the Centers for Disease Control and Prevention. Limited studies have genotyped Carbapenem-resistant Escherichia coli using whole genome sequencing to characterize the most common lineages and resistance and virulence genes. The aim of this study was to characterize sequence data from carbapenem-resistant E. coli isolates (n = 82) collected longitudinally by the Alameda County Public Health Laboratory (ACPHL) between 2017 and 2019. E. coli genomes were screened for antibiotic resistance genes (ARGs) and extraintestinal pathogenic E. coli virulence factor genes (VFGs). The carbapenem-resistant E. coli lineages were diverse, with 24 distinct sequence types (STs) represented, including clinically important STs: ST131, ST69, ST95, and ST73. All Ambler classes of Carbapenemases were present, with NDM-5 being most the frequently detected. Nearly all isolates (90%) contained genes encoding resistance to third-generation cephalosporins; blaCTX-M genes were most common. The number of virulence genes present within pandemic STs was significantly higher than the number in non-pandemic lineages (p = 0.035). Virulence genes fimA (92%), trat (71%), kpsM (54%), and iutA (46%) were the most prevalent within the isolates. Considering the public health risk associated with CRE, these data enhance our understanding of the diversity of clinically important E. coli that are circulating in Alameda County, California.

Detecting SARS-CoV-2 variants in wastewater and their correlation with circulating variants in the communities.

Detection of SARS-CoV-2 viral load in wastewater has been highly informative in estimating the approximate number of infected individuals in the surrounding communities. Recent developments in wastewater monitoring to determine community prevalence of COVID-19 further extends into identifying SARS-CoV-2 variants, including those being monitored for having enhanced transmissibility. We sequenced genomic RNA derived from wastewater to determine the variants of coronaviruses circulating in the communities. Wastewater samples were collected from Truckee Meadows Water Reclamation Facility (TMWRF) from November 2020 to June 2021. SARS-CoV-2 variants resulting from wastewater were compared with the variants detected in infected individuals' clinical specimens (nasal/nasopharyngeal swabs) during the same period and found conclusively in agreement. Therefore, wastewater monitoring for SARS-CoV-2 variants in the community is a feasible strategy as a complementary tool to clinical specimen testing in the latter's absence.

Rapid repeat infection of SARS-CoV-2 by two highly distinct delta-lineage viruses.

An instance of sequential infection of an individual with, firstly, the Delta variant and secondly a Delta-sub-lineage has been identified. The individual was found positive for the AY.26 lineage 22 days after being found positive for the Delta [B.1.617.2] variant. The viruses associated with the cases showed dramatic genomic difference, including 31 changes that resulted in deletions or amino acid substitutions. Seven of these differences were observed in the Spike protein. The patient in question was between 30 and 35 years old and had no underlying health conditions. Though singular, this case illustrates the possibility that infection with the Delta variant may not itself be fully protective against a population of SARS-CoV-2 variants that are becoming increasingly diverse.

Use of wastewater surveillance for early detection of Alpha and Epsilon SARS-CoV-2 variants of concern and estimation of overall COVID-19 infection burden.

A decline in diagnostic testing for SARS-CoV-2 is expected to delay the tracking of COVID-19 variants of concern and interest in the United States. We hypothesize that wastewater surveillance programs provide an effective alternative for detecting emerging variants and assessing COVID-19 incidence, particularly when clinical surveillance is limited. Here, we analyzed SARS-CoV-2 RNA in wastewater from eight locations across Southern Nevada between March 2020 and April 2021. Trends in SARS-CoV-2 RNA concentrations (ranging from 4.3 log10 gc/L to 8.7 log10 gc/L) matched trends in confirmed COVID-19 incidence, but wastewater surveillance also highlighted several limitations with the clinical data. Amplicon-based whole genome sequencing (WGS) of 86 wastewater samples identified the B.1.1.7 (Alpha) and B.1.429 (Epsilon) lineages in December 2020, but clinical sequencing failed to identify the variants until January 2021, thereby demonstrating that 'pooled' wastewater samples can sometimes expedite variant detection. Also, by calibrating fecal shedding (11.4 log10 gc/infection) and wastewater surveillance data to reported seroprevalence, we estimate that ~38% of individuals in Southern Nevada had been infected by SARS-CoV-2 as of April 2021, which is significantly higher than the 10% of individuals confirmed through clinical testing. Sewershed-specific ascertainment ratios (i.e., X-fold infection undercounts) ranged from 1.0 to 7.7, potentially due to demographic differences. Our data underscore the growing application of wastewater surveillance in not only the identification and quantification of infectious agents, but also the detection of variants of concern that may be missed when diagnostic testing is limited or unavailable.

Detecting SARS-CoV-2 Variants in Wastewater and Their Correlation With Circulating Variants in the Communities.

Detection of SARS-CoV-2 viral load in wastewater has been highly informative in estimating the approximate number of infected individuals in the surrounding communities. Recent developments in wastewater monitoring to determine community prevalence of COVID-19 further extends into identifying SARS-CoV-2 variants, including those being monitored for having enhanced transmissibility. We sequenced genomic RNA derived from wastewater to determine the variants of coronaviruses circulating in the communities. Wastewater samples were collected from Truckee Meadows Water Reclamation Facility (TMWRF) from November 2021 to June 2021 were analyzed for SARS-CoV-2 variants and were compared with the variants detected in the clinical specimens (nasal/nasopharyngeal swabs) of infected individuals during the same period. The comparison was found to be conclusively in agreement. Therefore, wastewater monitoring for SARS-CoV-2 variants in the community is a feasible strategy both as a complementary tool to clinical specimen testing and in the latter's absence.

Screening of SARS-CoV-2 antivirals through a cell-based RNA-dependent RNA polymerase (RdRp) reporter assay.

COVID-19 (Coronavirus Disease 2019) caused by SARS-CoV-2 (Severe Acute Respiratory Syndrome CoronaVirus-2) continues to pose an international public health threat and thus far, has resulted in greater than 6.4 million deaths worldwide. Vaccines are critical tools to limit COVID-19 spread, but antiviral drug development is an ongoing global priority due to fast-spreading COVID-19 variants that may elude vaccine efficacies. The RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 is an essential enzyme of viral replication and transcription machinery complex. Therefore, the RdRp is an attractive target for the development of effective anti-COVID-19 therapeutics. In this study, we developed a cell-based assay to determine the enzymatic activity of SARS-CoV-2 RdRp through a luciferase reporter system. The SARS-CoV-2 RdRp reporter assay was validated using known inhibitors of RdRp polymerase, remdesivir along with other anti-virals including ribavirin, penciclovir, rhoifolin, 5'CT, and dasabuvir. Dasabuvir (an FDA-approved drug) exhibited promising RdRp inhibitory activity among these inhibitors. Anti-viral activity of dasabuvir was also tested on the replication of SARS-CoV-2 through infection of Vero E6 cells. Dasabuvir inhibited the replication of SARS-CoV-2, USA-WA1/2020 as well as B.1.617.2 (delta variant) in Vero E6 cells in a dose-dependent manner with EC50 values 9.47 µM and 10.48 µM, for USA-WA1/2020 and B.1.617.2 variants, respectively. Our results suggest that dasabuvir can be further evaluated as a therapeutic drug for COVID-19. Importantly, this system provides a robust, target-specific, and high-throughput screening compatible (z- and z'-factors of >0.5) platforms that will be a valuable tool for screening SARS-CoV-2 RdRp inhibitors.

Imipenem-Relebactam Susceptibility and Genotypic Characteristics of Carbapenem-Resistant Enterobacterales Identified during Population-Based Surveillance.

Laboratories submit all carbapenem-resistant Enterobacter, Escherichia coli, and Klebsiella species to the Alameda County Public Health Department (ACPHD). ACPHD evaluated 75 isolates submitted during 9 months for susceptibility to imipenem-relebactam (I-R) and, using whole-genome sequencing, identified ß-lactamase genes. Of 60 (80%) isolates susceptible to I-R, 8 (13%) had detectable carbapenemase genes, including 4 KPC, two NDM, and two OXA-48-like; we described the relationship between the presence of ß-lactamase resistance genes and susceptibility to I-R.

Genomic surveillance of Nevada patients revealed prevalence of unique SARS-CoV-2 variants bearing mutations in the RdRp gene.

Patients with signs of COVID-19 were tested through diagnostic RT-PCR for SARS-CoV-2 using RNA extracted from the nasopharyngeal/nasal swabs. To determine the variants of SARS-CoV-2 circulating in the state of Nevada, specimens from 200 COVID-19 patients were sequenced through our robust sequencing platform, which enabled sequencing of SARS-CoV-2 from specimens with even very low viral loads, without the need of culture-based amplification. High genome coverage allowed the identification of single and multi-nucleotide variants in SARS-CoV-2 in the community and their phylogenetic relationships with other variants present during the same period of the outbreak. We report the occurrence of a novel mutation at 323aa (314aa of orf1b) of nsp12 (RNA-dependent RNA polymerase) changed to phenylalanine (F) from proline (P), in the first reported isolate of SARS-CoV-2, Wuhan-Hu-1. This 323F variant was present at a very high frequency in Northern Nevada. Structural modeling determined this mutation in the interface domain, which is important for the association of accessory proteins required for the polymerase. In conclusion, we report the introduction of specific SARS-CoV-2 variants at very high frequency in distinct geographic locations, which is important for understanding the evolution and circulation of SARS-CoV-2 variants of public health importance, while it circulates in humans.

Genomic evidence for reinfection with SARS-CoV-2: a case study.

BACKGROUND: The degree of protective immunity conferred by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently unknown. As such, the possibility of reinfection with SARS-CoV-2 is not well understood. We describe an investigation of two instances of SARS-CoV-2 infection in the same individual. METHODS: A 25-year-old man who was a resident of Washoe County in the US state of Nevada presented to health authorities on two occasions with symptoms of viral infection, once at a community testing event in April, 2020, and a second time to primary care then hospital at the end of May and beginning of June, 2020. Nasopharyngeal swabs were obtained from the patient at each presentation and twice during follow-up. Nucleic acid amplification testing was done to confirm SARS-CoV-2 infection. We did next-generation sequencing of SARS-CoV-2 extracted from nasopharyngeal swabs. Sequence data were assessed by two different bioinformatic methodologies. A short tandem repeat marker was used for fragment analysis to confirm that samples from both infections came from the same individual. FINDINGS: The patient had two positive tests for SARS-CoV-2, the first on April 18, 2020, and the second on June 5, 2020, separated by two negative tests done during follow-up in May, 2020. Genomic analysis of SARS-CoV-2 showed genetically significant differences between each variant associated with each instance of infection. The second infection was symptomatically more severe than the first. INTERPRETATION: Genetic discordance of the two SARS-CoV-2 specimens was greater than could be accounted for by short-term in vivo evolution. These findings suggest that the patient was infected by SARS-CoV-2 on two separate occasions by a genetically distinct virus. Thus, previous exposure to SARS-CoV-2 might not guarantee total immunity in all cases. All individuals, whether previously diagnosed with COVID-19 or not, should take identical precautions to avoid infection with SARS-CoV-2. The implications of reinfections could be relevant for vaccine development and application. FUNDING: Nevada IDEA Network of Biomedical Research, and the National Institute of General Medical Sciences (National Institutes of Health).

Characteristics of viral specimens collected from asymptomatic and fatal cases of COVID-19.

We sought to determine the characteristics of viral specimens associated with fatal cases, asymptomatic cases and non-fatal symptomatic cases of COVID-19. This included the analysis of 1264 specimens found reactive for at least two SARS-CoV-2 specific loci from people screened for infection in Northern Nevada in March-May of 2020. Of these, 30 were specimens from fatal cases, while 23 were from positive, asymptomatic cases. We assessed the relative amounts of SARS-CoV-2 RNA from sample swabs by real-time PCR and use of the threshold crossing value (Ct). Moreover, we compared the amount of human RNase P found on the same swabs. A considerably higher viral load was found to be associated with swabs from cases involving fatality and the difference was found to be strongly statistically significant. Noting this difference, we sought to assess whether any genetic correlation could be found in association with virus from fatal cases using whole genome sequencing. While no common genetic elements were discerned, one branch of epidemiologically linked fatal cases did have two point mutations, which no other of 156 sequenced cases from northern Nevada had. The mutations caused amino acid changes in the 3'-5' exonuclease protein, and the product of the gene, orf8.

Genomic surveillance of Nevada patients revealed prevalence of unique SARS-CoV-2 variants bearing mutations in the RdRp gene.

Patients with signs of COVID-19 were tested with CDC approved diagnostic RT-PCR for SARS-CoV-2 using RNA extracted from nasopharyngeal/nasal swabs. In order to determine the variants of SARS-CoV-2 circulating in the state of Nevada, 200 patient specimens from COVID-19 patients were sequenced through our robust protocol for sequencing SARS-CoV-2 genomes. Our protocol enabled sequencing of SARS-CoV-2 genome directly from the specimens, with even very low viral loads, without the need of culture-based amplification. This allowed the identification of specific nucleotide variants including those coding for D614G and clades defining mutations. These sequences were further analyzed for determining SARS-CoV-2 variants circulating in the state of Nevada and their phylogenetic relationships with other variants present in the united states and the world during the same period of the outbreak. Our study reports the occurrence of a novel variant in the nsp12 (RNA dependent RNA Polymerase) protein at residue 323 (314aa of orf1b) to Phenylalanine (F) from Proline (P), present in the original isolate of SARS-CoV-2 (Wuhan-Hu-1). This 323F variant is found at a very high frequency (46% of the tested specimen) in Northern Nevada. Functional significance of this unique and highly prevalent variant of SARS-CoV-2 with RdRp mutation is currently under investigation but structural modeling showed this 323aa residue in the interface domain of RdRp, which is required for association with accessory proteins. In conclusion, we report the introduction of specific SARS-CoV-2 variants at a very high frequency within a distinct geographic location, which is important for clinical and public health perspectives in understanding the evolution of SARS-CoV-2 while in circulation.

F Plasmids Are the Major Carriers of Antibiotic Resistance Genes in Human-Associated Commensal Escherichia coli.

The evolution and propagation of antibiotic resistance by bacterial pathogens are significant threats to global public health. Contemporary DNA sequencing tools were applied here to gain insight into carriage of antibiotic resistance genes in Escherichia coli, a ubiquitous commensal bacterium in the gut microbiome in humans and many animals, and a common pathogen. Draft genome sequences generated for a collection of 101 E. coli strains isolated from healthy undergraduate students showed that horizontally acquired antibiotic resistance genes accounted for most resistance phenotypes, the primary exception being resistance to quinolones due to chromosomal mutations. A subset of 29 diverse isolates carrying acquired resistance genes and 21 control isolates lacking such genes were further subjected to long-read DNA sequencing to enable complete or nearly complete genome assembly. Acquired resistance genes primarily resided on F plasmids (101/153 [67%]), with smaller numbers on chromosomes (30/153 [20%]), IncI complex plasmids (15/153 [10%]), and small mobilizable plasmids (5/153 [3%]). Nearly all resistance genes were found in the context of known transposable elements. Very few structurally conserved plasmids with antibiotic resistance genes were identified, with the exception of an ∼90-kb F plasmid in sequence type 1193 (ST1193) isolates that appears to serve as a platform for resistance genes and may have virulence-related functions as well. Carriage of antibiotic resistance genes on transposable elements and mobile plasmids in commensal E. coli renders the resistome highly dynamic.IMPORTANCE Rising antibiotic resistance in human-associated bacterial pathogens is a serious threat to our ability to treat many infectious diseases. It is critical to understand how acquired resistance genes move in and through bacteria associated with humans, particularly for species such as Escherichia coli that are very common in the human gut but can also be dangerous pathogens. This work combined two distinct DNA sequencing approaches to allow us to explore the genomes of E. coli from college students to show that the antibiotic resistance genes these bacteria have acquired are usually carried on a specific type of plasmid that is naturally transferrable to other E. coli, and likely to other related bacteria.

Sensitive and Specific Detection of SARS-CoV-2 Antibodies Using a High-Throughput, Fully Automated Liquid-Handling Robotic System.

As of July 22, 2020, more than 14.7 million infections of SARS-CoV-2, the virus responsible for Coronavirus Disease 2019 (COVID-19), have been confirmed globally. Serological assays are essential for community screening, assessing infection prevalence, aiding identification of infected patients, and enacting appropriate treatment and quarantine protocols in the battle against this rapidly expanding pandemic. Antibody detection by agglutination-PCR (ADAP) is a pure solution phase immunoassay that generates a PCR amplifiable signal when patient antibodies agglutinate DNA-barcoded antigen probes into a dense immune complex. Here, we present an ultrasensitive and high-throughput automated liquid biopsy assay based on the Hamilton Microlab ADAP STAR automated liquid-handling platform, which was developed and validated for the qualitative detection of total antibodies against spike protein 1 (S1) of SARS-CoV-2 that uses as little as 4 µL of serum. To assess the clinical performance of the ADAP assay, 57 PCR-confirmed COVID-19 patients and 223 control patients were tested. The assay showed a sensitivity of 98% (56/57) and a specificity of 99.55% (222/223). Notably, the SARS-CoV-2-negative control patients included individuals with other common coronaviral infections, such as CoV-NL63 and CoV-HKU, which did not cross-react. In addition to high performance, the hands-free automated workstation enabled high-throughput sample processing to reduce screening workload while helping to minimize analyst contact with biohazardous samples. Therefore, the ADAP STAR liquid-handling workstation can be used as a valuable tool to address the COVID-19 global pandemic.