Supplementation with soluble or insoluble rice-bran fibers increases short-chain fatty acid producing bacteria in the gut microbiota in vitro.

Introduction: Studies have shown that a diet high in fiber and prebiotics has a positive impact on human health due largely to the fermentation of these compounds by the gut microbiota. One underutilized source of fiber may be rice bran, a waste product of rice processing that is used most frequently as an additive to livestock feed but may be a good source of fibers and other phenolic compounds as a human diet supplement. Previous studies focused on specific compounds extracted from rice bran showed that soluble fibers extracted from rice bran can improve glucose response and reduce weight gain in mouse models. However, less is known about changes in the human gut microbiota in response to regular rice bran consumption. Methods: In this study, we used a Simulator of the Human Intestinal Microbial Ecology (SHIME®) to cultivate the human gut microbiota of 3 different donors in conditions containing either soluble or insoluble fiber fractions from rice bran. Using 16S rRNA amplicon sequencing and targeted metabolomics via Gas Chromatography-Mass Spectrometry, we explored how gut microbial communities developed provided different supplemental fiber sources. Results: We found that insoluble and soluble fiber fractions increased short-chain fatty acid production, indicating that both fractions were fermented. However, there were differences in response between donors, for example the gut microbiota from donor 1 increased acetic acid production with both fiber types compared with control; whereas for donors 2 and 3, butanoic acid production increased with ISF and SF supplementation. Both soluble and insoluble rice bran fractions increased the abundance of Bifidobacterium and Lachnospiraceae taxa. Discussion: Overall, analysis of the effect of soluble and insoluble rice bran fractions on the human in vitro gut microbiota and the metabolites produced revealed individually variant responses to these prebiotics.

Liberation of host heme by Clostridioides difficile-mediated damage enhances Enterococcus faecalis fitness during infection.

IMPORTANCE: Clostridioides difficile and Enterococcus faecalis are two pathogens of great public health importance. Both bacteria colonize the human gastrointestinal tract where they are known to interact in ways that worsen disease outcomes. We show that the damage associated with C. difficile infection (CDI) releases nutrients that benefit E. faecalis. One particular nutrient, heme, allows E. faecalis to use oxygen to generate energy and grow better in the gut. Understanding the mechanisms of these interspecies interactions could inform therapeutic strategies for CDI.

RdJ detection tests to identify a unique MRSA clone of ST105-SCCmecII lineage and its variants disseminated in the metropolitan region of Rio de Janeiro.

Hospital bloodstream infection (BSI) caused by methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of morbidity and mortality and is frequently related to invasive procedures and medically complex patients. An important feature of MRSA is the clonal structure of its population. Specific MRSA clones may differ in their pathogenic, epidemiological, and antimicrobial resistance profiles. Whole-genome sequencing is currently the most robust and discriminatory technique for tracking hypervirulent/well-adapted MRSA clones. However, it remains an expensive and time-consuming technique that requires specialized personnel. In this work, we describe a pangenome protocol, based on binary matrix (1,0) of open reading frames (ORFs), that can be used to quickly find diagnostic, apomorphic sequence mutations that can serve as biomarkers. We use this technique to create a diagnostic screen for MRSA isolates circulating in the Rio de Janeiro metropolitan area, the RdJ clone, which is prevalent in BSI. The method described here has 100% specificity and sensitivity, eliminating the need to use genomic sequencing for clonal identification. The protocol used is relatively simple and all the steps, formulas and commands used are described in this work, such that this strategy can also be used to identify other MRSA clones and even clones from other bacterial species.

Impact of Ivermectin on the Gut Microbial Ecosystem.

Ivermectin is a an anti-helminthic that is critical globally for both human and veterinary care. To the best of our knowledge, information available regarding the influence of ivermectin (IVM) on the gut microbiota has only been collected from diseased donors, who were treated with IVM alone or in combination with other medicines. Results thus obtained were influenced by multiple elements beyond IVM, such as disease, and other medical treatments. The research presented here investigated the impact of IVM on the gut microbial structure established in a Triple-SHIME® (simulator of the human intestinal microbial ecosystem), using fecal material from three healthy adults. The microbial communities were grown using three different culture media: standard SHIME media and SHIME media with either soluble or insoluble fiber added (control, SF, ISF). IVM introduced minor and temporary changes to the gut microbial community in terms of composition and metabolite production, as revealed by 16S rRNA amplicon sequencing analysis, flow cytometry, and GC-MS. Thus, it was concluded that IVM is not expected to induce dysbiosis or yield adverse effects if administered to healthy adults. In addition, the donor's starting community influences the relationship between IVM and the gut microbiome, and the soluble fiber component in feed could protect the gut microbiota from IVM; an increase in short-chain fatty acid production was predicted by PICRUSt2 and detected with IVM treatment.

Parallel evolution of linezolid-resistant Staphylococcus aureus in patients with cystic fibrosis.

Linezolid is an antibiotic used to treat serious Staphylococcus aureus infections. Resistance to linezolid is considered rare but could emerge with repeated dosing. We recently reported widespread prescription of linezolid for a cohort of patients with cystic fibrosis (CF). The goals of this study were to determine the incidence of linezolid-resistant methicillin-resistant Staphylococcus aureus (MRSA) in CF and identify molecular mechanisms for linezolid resistance. We identified patients who cultured S. aureus resistant to linezolid with minimum inhibitory concentration (MIC) >4 at the University of Iowa CF Center between 2008 and 2018. We obtained isolates from these patients and retested susceptibility to linezolid using broth microdilution. We used whole genome sequencing to perform phylogenetic analysis of linezolid-resistant isolates and examine sequences for mutations or accessory genes that confer linezolid resistance. Between 2008 and 2018, 111 patients received linezolid, and 4 of these patients cultured linezolid-resistant S. aureus. We sequenced 11 resistant and 21 susceptible isolates from these 4 subjects. Phylogenetic analysis indicated that linezolid resistance developed in ST5 or ST105 backgrounds. Three individuals had linezolid-resistant S. aureus with a G2576T mutation in 23S rRNA. One of these subjects additionally had a mutS- mutL- hypermutating S. aureus that produced five resistant isolates with multiple ribosomal subunit mutations. In one subject, the genetic basis for linezolid resistance was unclear. We conclude that linezolid resistant S. aureus can occur through multiple genetic mechanisms in patients with repeated exposure to this antibiotic. IMPORTANCE Patients with cystic fibrosis have persistent lung infections with Staphylococcus aureus that require extensive antibiotic treatments. Linezolid, an antibiotic given by oral or intravenous route, is prescribed repeatedly for patients whose lung disease has progressed. After treatment with linezolid, S. aureus strains can evolve antibiotic resistance through multiple genetic mechanisms. In addition to a common mutation in the 23S ribosomal RNA known to confer linezolid resistance, S. aureus strains can evolve novel resistance based on a combination of mutations affecting the bacterial ribosome. This combination of mutations was observed in a strain that exhibited hypermutation owing to the loss of the DNA repair genes mutS and mutL. In this cohort of patients with cystic fibrosis, linezolid resistance was transient, possibly due to the growth disadvantage of resistant strains. However, ongoing chronic exposure to linezolid may create optimal conditions for the future emergence of resistance to this critical antibiotic.

Dynamics of the MRSA Population in a Chilean Hospital: a Phylogenomic Analysis (2000-2016).

The global dissemination of methicillin-resistant Staphylococcus aureus (MRSA) is associated with the emergence and establishment of clones in specific geographic areas. The Chilean-Cordobes clone (ChC) (ST5-SCCmecI) has been the predominant MRSA clone in Chile since its first description in 1998, despite the report of other emerging MRSA clones in recent years. Here, we characterize the evolutionary history of MRSA from 2000 to 2016 in a Chilean tertiary health care center using phylogenomic analyses. We sequenced 469 MRSA isolates collected between 2000 and 2016. We evaluated the temporal trends of the circulating clones and performed a phylogenomic reconstruction to characterize the clonal dynamics. We found a significant increase in the diversity and richness of sequence types (STs; Spearman r = 0.8748, P < 0.0001) with a Shannon diversity index increasing from 0.221 in the year 2000 to 1.33 in 2016, and an effective diversity (Hill number; q = 2) increasing from 1.12 to 2.71. The temporal trend analysis revealed that in the period 2000 to 2003 most of the isolates (94.2%; n = 98) belonged to the ChC clone. However, since then, the frequency of the ChC clone has decreased over time, accounting for 52% of the collection in the 2013 to 2016 period. This decline was accompanied by the rise of two emerging MRSA lineages, ST105-SCCmecII and ST72-SCCmecVI. In conclusion, the ChC clone remains the most frequent MRSA lineage, but this lineage is gradually being replaced by several emerging clones, the most important of which is clone ST105-SCCmecII. To the best of our knowledge, this is the largest study of MRSA clonal dynamics performed in South America. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) is a major public health pathogen that disseminates through the emergence of successful dominant clones in specific geographic regions. Knowledge of the dissemination and molecular epidemiology of MRSA in Latin America is scarce and is largely based on small studies or more limited typing techniques that lack the resolution to represent an accurate description of the genomic landscape. We used whole-genome sequencing to study 469 MRSA isolates collected between 2000 and 2016 in Chile providing the largest and most detailed study of clonal dynamics of MRSA in South America to date. We found a significant increase in the diversity of MRSA clones circulating over the 17-year study period. Additionally, we describe the emergence of two novel clones (ST105-SCCmecII and ST72-SCCmecVI), which have been gradually increasing in frequency over time. Our results drastically improve our understanding of the dissemination and update our knowledge about MRSA in Latin America.

Heavy Metal Pollution From a Major Earthquake and Tsunami in Chile Is Associated With Geographic Divergence of Clinical Isolates of Methicillin-Resistant Staphylococcus aureus in Latin America.

Methicillin-resistant Staphylococcus aureus (MRSA) is a priority pathogen listed by the World Health Organization. The global spread of MRSA is characterized by successive waves of epidemic clones that predominate in specific geographical regions. The acquisition of genes encoding resistance to heavy-metals is thought to be a key feature in the divergence and geographical spread of MRSA. Increasing evidence suggests that extreme natural events, such as earthquakes and tsunamis, could release heavy-metals into the environment. However, the impact of environmental exposition to heavy-metals on the divergence and spread of MRSA clones has been insufficiently explored. We assess the association between a major earthquake and tsunami in an industrialized port in southern Chile and MRSA clone divergence in Latin America. We performed a phylogenomic reconstruction of 113 MRSA clinical isolates from seven Latin American healthcare centers, including 25 isolates collected in a geographic area affected by an earthquake and tsunami that led to high levels of heavy-metal environmental contamination. We found a divergence event strongly associated with the presence of a plasmid harboring heavy-metal resistance genes in the isolates obtained in the area where the earthquake and tsunami occurred. Moreover, clinical isolates carrying this plasmid showed increased tolerance to mercury, arsenic, and cadmium. We also observed a physiological burden in the plasmid-carrying isolates in absence of heavy-metals. Our results are the first evidence that suggests that heavy-metal contamination, in the aftermath of an environmental disaster, appears to be a key evolutionary event for the spread and dissemination of MRSA in Latin America.

Parallel Evolution of Linezolid Resistant Staphylococcus aureus in Patients with Cystic Fibrosis.

Background: Linezolid is an antibiotic used to treat serious Staphylococcus aureus infections. Resistance to linezolid is considered rare but could emerge with repeated dosing. We recently reported widespread prescription of linezolid for a cohort of patients with cystic fibrosis (CF). Objectives: The goals of this study were to determine the incidence of linezolid resistance in CF and identify molecular mechanisms for linezolid resistance. Methods: We identified patients with S. aureus resistant to linezolid (MIC > 4) at the University of Iowa CF Center between 2008 and 2018. We obtained isolates from these patients and retested susceptibility to linezolid using broth microdilution. We used whole genome sequencing to perform phylogenetic analysis of linezolid resistant isolates and examine sequences for mutations or accessory genes that confer linezolid resistance. Main Results: Between 2008 and 2018, 111 patients received linezolid and 4 of these patients cultured linezolid resistant S. aureus . We sequenced 11 resistant and 21 susceptible isolates from these 4 subjects. Phylogenetic analysis indicated that linezolid resistance developed in ST5 or ST105 backgrounds. Three individuals had linezolid resistant S. aureus with a G2576T mutation in 23S rRNA. One of these subjects additionally had a mutS - mutL - hypermutating S. aureus that produced 5 resistant isolates with multiple ribosomal subunit mutations. In one subject, the genetic basis for linezolid resistance was unclear. Conclusions: Linezolid resistance evolved in 4 of 111 patients in this study. Linezolid resistance occurred by multiple genetic mechanisms. All resistant strains developed in ST5 or ST105 MRSA backgrounds. Key Point: Linezolid resistance arises through multiple genetic mechanisms and could be facilitated by mutator phenotypes. Linezolid resistance was transient, possibly due to growth disadvantage.

Novel Tetrahydro-[1,2,4]triazolo[3,4-a]isoquinoline Chalcones Suppress Breast Carcinoma through Cell Cycle Arrests and Apoptosis.

Chalcones are interesting anticancer drug candidates which have attracted much interest due to their unique structure and their extensive biological activity. Various functional modifications in chalcones have been reported, along with their pharmacological properties. In the current study, novel chalcone derivatives with the chemical base of tetrahydro-[1,2,4]triazolo[3,4-a]isoquinolin-3-yl)-3-arylprop-2-en-1-one were synthesized, and the structure of their molecules was confirmed through NMR spectroscopy. The antitumor activity of these newly synthesized chalcone derivatives was tested on mouse (Luc-4T1) and human (MDA-MB-231) breast cancer cell lines. The antiproliferative effect was evaluated through SRB screening and the MTT assay after 48 h of treatment at different concentrations. Interestingly, among the tested chalcone derivatives, chalcone analogues with a methoxy group were found to have significant anticancer activity and displayed gradient-dependent inhibition against breast cancer cell proliferation. The anticancer properties of these unique analogues were examined further by cytometric analysis of the cell cycle, quantitative PCR, and the caspases-Glo 3/7 assay. Chalcone methoxy derivatives showed the capability of cell cycle arrest and increased Bax/Bcl2 mRNA ratios as well as caspases 3/7 activity. The molecular docking analysis suggests that these chalcone methoxy derivatives may inhibit anti-apoptotic proteins, particularly cIAP1, BCL2, and EGFRK proteins. In conclusion, our findings confirm that chalcone methoxy derivatives could be considered to be potent drug candidates against breast cancer.

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

Introduction: 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 epidemic (SAE) clade and North American epidemic (NAE) clade. In addition, historical isolates collected prior to the emergence of epidemics can help reconstruct early events in the history of this lineage. Methods: 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. Results: Phylogenetic analysis with these genomes confirms that the NAE and SAE 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 provide evidence for a North American origin of the USA300 lineage and identify multiple introductions of USA300 into South and 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 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.

Fructooligosaccharides (FOS) differentially modifies the in vitro gut microbiota in an age-dependent manner.

Introduction: Fructooligosaccharides (FOS) are well-known carbohydrates that promote healthy gut microbiota and have been previously demonstrated to enhance levels of Bifidobacterium and Lactobacillus. Its bifidogenic properties are associated with positive health outcomes such as reduced obesity and anti-inflammatory properties, and, therefore, is in use as a prebiotic supplement to support healthy gut microbiota. However, the gut microbiota changes with age, which may lead to differential responses to treatments with prebiotics and other dietary supplements. Methods: To address this concern, we implemented a 24-h in vitro culturing method to determine whether FOS treatment in three different adult age groups would have a differential effect. The age groups of interest ranged from 25 to 70 years and were split into young adults, adults, and older adults for the purposes of this analysis. Metagenomics and short-chain fatty acid analysis were performed to determine changes in the structure and function of the microbial communities. Results: These analyses found that FOS created a bifidogenic response in all age groups, increased overall SCFA levels, decreased alpha diversity, and shifted the communities to be more similar in beta diversity metrics. However, the age groups differed in which taxa were most prevalent or most affected by FOS treatment. Discussion: Overall, the results of this study demonstrate the positive effects of FOS on the gut microbiome, and importantly, how age may play a role in the effectiveness of this prebiotic.

Multidrug-Resistant Methicillin-Resistant Staphylococcus aureus Associated with Bacteremia and Monocyte Evasion, Rio de Janeiro, Brazil.

We typed 600 methicillin-resistant Staphylococcus aureus (MRSA) isolates collected in 51 hospitals in the Rio de Janeiro, Brazil, metropolitan area during 2014-2017. We found that multiple new clonal complex (CC) 5 sequence types had replaced previously dominant MRSA lineages in hospitals. Whole-genome analysis of 208 isolates revealed an emerging sublineage of multidrug-resistant MRSA, sequence type 105, staphylococcal cassette chromosome mec II, spa t002, which we designated the Rio de Janeiro (RdJ) clone. Using molecular clock analysis, we hypothesized that this lineage began to expand in the Rio de Janeiro metropolitan area in 2009. Multivariate analysis supported an association between bloodstream infections and the CC5 lineage that includes the RdJ clone. Compared with other closely related isolates, representative isolates of the RdJ clone more effectively evaded immune function related to monocytic cells, as evidenced by decreased phagocytosis rate and increased numbers of viable unphagocytosed (free) bacteria after in vitro exposure to monocytes.

SARS-CoV-2 variants associated with vaccine breakthrough in the Delaware Valley through summer 2021.

The severe acute respiratory coronavirus-2 (SARS-CoV-2) is the cause of the global outbreak of COVID-19. Evidence suggests that the virus is evolving to allow efficient spread through the human population, including vaccinated individuals. Here we report a study of viral variants from surveillance of the Delaware Valley, including the city of Philadelphia, and variants infecting vaccinated subjects. We sequenced and analyzed complete viral genomes from 2621 surveillance samples from March 2020 to September 2021 and compared them to genome sequences from 159 vaccine breakthroughs. In the early spring of 2020, all detected variants were of the B.1 and closely related lineages. A mixture of lineages followed, notably including B.1.243 followed by B.1.1.7 (alpha), with other lineages present at lower levels. Later isolations were dominated by B.1.617.2 (delta) and other delta lineages; delta was the exclusive variant present by the last time sampled. To investigate whether any variants appeared preferentially in vaccine breakthroughs, we devised a model based on Bayesian autoregressive moving average logistic multinomial regression to allow rigorous comparison. This revealed that B.1.617.2 (delta) showed three-fold enrichment in vaccine breakthrough cases (odds ratio of 3; 95% credible interval 0.89-11). Viral point substitutions could also be associated with vaccine breakthroughs, notably the N501Y substitution found in the alpha, beta and gamma variants (odds ratio 2.04; 95% credible interval of 1.25-3.18). This study thus provides a detailed picture of viral evolution in the Delaware Valley and a geographically matched analysis of vaccine breakthroughs; it also introduces a rigorous statistical approach to interrogating enrichment of viral variants.

Copper Resistance Promotes Fitness of Methicillin-Resistant Staphylococcus aureus during Urinary Tract Infection.

Urinary tract infection (UTI) is one of the most common infectious conditions affecting people in the United States and around the world. Our knowledge of the host-pathogen interaction during UTI caused by Gram-positive bacterial uropathogens is limited compared to that for Gram-negative pathogens. Here, we investigated whether copper and the primary copper-containing protein, ceruloplasmin, are mobilized to urine during naturally occurring UTI caused by Gram-positive uropathogens in patients. Next, we probed the role of copper resistance in the fitness of methicillin-resistant Staphylococcus aureus (MRSA) during experimental UTI in a murine model. Our findings demonstrate that urinary copper and ceruloplasmin content are elevated during UTI caused by Enterococcus faecalis, S. aureus, S. epidermidis, and S. saprophyticus. MRSA strains successfully colonize the urinary tract of female CBA mice with selective induction of inflammation in the kidneys but not the bladder. MRSA mutants lacking CopL, a copper-binding cell surface lipoprotein, and the ACME genomic region containing copL, exhibit decreased fitness in the mouse urinary tract compared to parental strains. Copper sensitivity assays, cell-associated copper and iron content, and bioavailability of iron during copper stress demonstrate that homeostasis of copper and iron is interlinked in S. aureus. Importantly, relative fitness of the MRSA mutant lacking the ACME region is further decreased in mice that receive supplemental copper compared to the parental strain. In summary, copper is mobilized to the urinary tract during UTI caused by Gram-positive pathogens, and copper resistance is a fitness factor for MRSA during UTI. IMPORTANCE Urinary tract infection (UTI) is an extremely common infectious condition affecting people throughout the world. Increasing antibiotic resistance in pathogens causing UTI threatens our ability to continue to treat patients in the clinics. Better understanding of the host-pathogen interface is critical for development of novel interventional strategies. Here, we sought to elucidate the role of copper in host-Staphylococcus aureus interaction during UTI. Our results reveal that copper is mobilized to the urine as a host response in patients with UTI. Our findings from the murine model of UTI demonstrate that copper resistance is involved in the fitness of methicillin-resistant S. aureus (MRSA) during interaction with the host. We also establish a critical link between adaptation to copper stress and iron homeostasis in S. aureus.

Jumping a Moving Train: SARS-CoV-2 Evolution in Real Time.

The field of molecular epidemiology responded to the SARS-CoV-2 pandemic with an unrivaled amount of whole viral genome sequencing. By the time this sentence is published we will have well surpassed 1.5 million whole genomes, more than 4 times the number of all microbial whole genomes deposited in GenBank and 35 times the total number of viral genomes. This extraordinary dataset that accrued in near real time has also given us an opportunity to chart the global and local evolution of a virus as it moves through the world population. The data itself presents challenges that have never been dealt with in molecular epidemiology, and tracking a virus that is changing so rapidly means that we are often running to catch up. Here we review what is known about the evolution of the virus, and the critical impact that whole genomes have had on our ability to trace back and track forward the spread of lineages of SARS-CoV-2. We then review what whole genomes have told us about basic biological properties of the virus such as transmissibility, virulence, and immune escape with a special emphasis on pediatric disease. We couch this discussion within the framework of systematic biology and phylogenetics, disciplines that have proven their worth again and again for identifying and deciphering the spread of epidemics, though they were largely developed in areas far removed from infectious disease and medicine.

Emerging SARS-CoV-2 Diversity Revealed by Rapid Whole-Genome Sequence Typing.

Discrete classification of SARS-CoV-2 viral genotypes can identify emerging strains and detect geographic spread, viral diversity, and transmission events. We developed a tool (GNU-based Virus IDentification [GNUVID]) that integrates whole-genome multilocus sequence typing and a supervised machine learning random forest-based classifier. We used GNUVID to assign sequence type (ST) profiles to all high-quality genomes available from GISAID. STs were clustered into clonal complexes (CCs) and then used to train a machine learning classifier. We used this tool to detect potential introduction and exportation events and to estimate effective viral diversity across locations and over time in 16 US states. GNUVID is a highly scalable tool for viral genotype classification (https://github.com/ahmedmagds/GNUVID) that can quickly classify hundreds of thousands of genomes in a way that is consistent with phylogeny. Our genotyping ST/CC analysis uncovered dynamic local changes in ST/CC prevalence and diversity with multiple replacement events in different states, an average of 20.6 putative introductions and 7.5 exportations for each state over the time period analyzed. We introduce the use of effective diversity metrics (Hill numbers) that can be used to estimate the impact of interventions (e.g., travel restrictions, vaccine uptake, mask mandates) on the variation in circulating viruses. Our classification tool uncovered multiple introduction and exportation events, as well as waves of expansion and replacement of SARS-CoV-2 genotypes in different states. GNUVID classification lends itself to measures of ecological diversity, and, with systematic genomic sampling, it could be used to track circulating viral diversity and identify emerging clones and hotspots.

Structure-guided microbial targeting of antistaphylococcal prodrugs.

Carboxy ester prodrugs are widely employed to increase oral absorption and potency of phosphonate antibiotics. Prodrugging can mask problematic chemical features that prevent cellular uptake and may enable tissue-specific compound delivery. However, many carboxy ester promoieties are rapidly hydrolyzed by serum esterases, limiting their therapeutic potential. While carboxy ester-based prodrug targeting is feasible, it has seen limited use in microbes as microbial esterase-specific promoieties have not been described. Here we identify the bacterial esterases, GloB and FrmB, that activate carboxy ester prodrugs in Staphylococcus aureus. Additionally, we determine the substrate specificities for FrmB and GloB and demonstrate the structural basis of these preferences. Finally, we establish the carboxy ester substrate specificities of human and mouse sera, ultimately identifying several promoieties likely to be serum esterase-resistant and microbially labile. These studies will enable structure-guided design of antistaphylococcal promoieties and expand the range of molecules to target staphylococcal pathogens.

Comparative Analysis of Emerging B.1.1.7+E484K SARS-CoV-2 Isolates.

We report the genome of a B.1.1.7+E484K severe acute respiratory syndrome coronavirus 2 from Southeastern Pennsylvania and compare it with all high-coverage B.1.1.7+E484K genomes (n = 235) available. Analyses showed the existence of at least 4 distinct clades of this variant circulating in the United States and the possibility of at least 59 independent acquisitions of the E484K mutation.

Comparative Analysis of Emerging B.1.1.7+E484K SARS-CoV-2 isolates from Pennsylvania.

Rapid whole genome sequencing of SARS-CoV-2 has presented the ability to detect new emerging variants of concern in near real time. Here we report the genome of a virus isolated in Pennsylvania in March 2021 that was identified as lineage B.1.1.7 (VOC-202012/01) that also harbors the E484K spike mutation, which has been shown to promote "escape" from neutralizing antibodies in vitro. We compare this sequence to the only 5 other B.1.1.7+E484K genomes from Pennsylvania, all of which were isolated in mid March. Beginning in February 2021, only a small number (n=60) of isolates with this profile have been detected in the US, and only a total of 253 have been reported globally (first in the UK in December 2020). Comparative genomics of all currently available high coverage B.1.1.7+E484K genomes (n=235) available on GISAID suggested the existence of 7 distinct groups or clonal complexes (CC; as defined by GNUVID) bearing the E484K mutation raising the possibility of 7 independent acquisitions of the E484K spike mutation in each background. Phylogenetic analysis suggested the presence of at least 3 distinct clades of B.1.1.7+E484K circulating in the US, with the Pennsylvanian isolates belonging to two distinct clades. Increased genomic surveillance will be crucial for detection of emerging variants of concern that can escape natural and vaccine induced immunity.