Biology subjects

Moustafa, A. M.

Publications and source records attributed to Moustafa, A. M..

11 recordsLinked to original sources

bioRxiv2026

Multi-lineage emergence of USA300 MRSA in in Chile and a potential role for environmental copper resistance

Methicillin-resistant S. aureus (MRSA) is prevalent worldwide, but individual strains of MRSA tend to spread and expand in relatively confined geographic areas. The USA300 lineage of MRSA is most well-known for its rapid epidemic spread in North America during the first decade of the 2000's leading to an overall increase in serious skin infections and the emergence of methicillin resistance in the majority of community acquired S. aureus infections. A distinct but closely related South American epidemic clade of MRSA USA300 (USA300-SAE), first reported in 2006, proliferated rapidly in Northern South America but was rarely found in other Latin American countries. In a new survey of 274 invasive MRSA isolates from 2022 collected in Chile, we saw a marked increase in USA300-SAE in Southern South America. We found that Chilean USA300-SAE is phylogenetically diverse and was likely introduced multiple times from multiple different South American countries over the course of more than a decade, but only recently became a highly prevalent clone. Using novel molecular diagnostic methods for isolates collected in 2023, we estimate that USA300-SAE made up more than 22% of invasive MRSA strains in Chile in that year and is likely still increasing. We show that copper-associated genes in the signature copper and mercury resistance mobile element (COMER) of USA300-SAE are strongly conserved, and that isogenic mutants of the copper resistance genes in COMER are highly susceptible to copper killing at concentrations seen in tap water. These findings suggest that environmental exposure to copper may provide a fitness advantage that enhances spread of this lineage.

microbiology↗

bioRxiv2025

Taxonomy-free fecal microbiome profiles enable robust prediction of immunotherapy response and toxicity in melanoma

The gut microbiome has been causally linked to the efficacy of immune-checkpoint inhibitor therapy (ICI), prompting numerous clinical trials of microbiome-targeting strategies. Yet, mechanisms by which gut microbiota shape immune responses remain elusive as taxonomic biomarkers have failed to generalize across multiple cohorts. In this study, we develop a taxonomy-agnostic framework to identify microbial biomarkers of ICI response and immune-related adverse event (irAE) occurrence from metagenomic sequencing. Applying this approach to four independent melanoma cohorts from clinical centers across the United States, we uncover gut microbial proteins produced by diverse bacterial taxa that consistently predict ICI response. Notably, we uncover a previously uncharacterized operon involved in cellular redox homeostasis that is encoded by different bacteria and reliably predicts irAE occurrence. We further validated the predictive power of this operon in a prospectively sequenced melanoma cohort. Our results demonstrate that taxa-agnostic microbial protein biomarkers are robust, generalizable, and provide a path towards pretreatment risk stratification for melanoma patients initiating ICI therapy.

cancer biology↗

bioRxiv2025

Optimizing methods for virome analysis based on studies of a synthetic viral community

Studies of whole viral populations--the "virome"--are yielding exciting new insights into biological systems, but methods are still being optimized. Here we describe generation and use of a synthetic viral community to assess several technical challenges important in virome analysis. Our mock community was comprised of phages lambda, T4, M13, MS2, and phi6, together with adeno-associated virus (AAV), murine hepatitis virus (MHV), and vaccinia virus (VV). We spiked the mock community into different human sample types, including stool, saliva, oropharyngeal (OP) wash, and bronchoalveolar lavage (BAL), then passed the samples through different virus enrichment protocols and analyzed by Illumina sequencing. Compared to direct metagenomic sequencing, VLP enrichment protocols greatly increased viral read yields from virus-rich samples such as from stool and saliva. Three VLP enrichment work flows were compared, and each was found to have strengths and weaknesses. Four methods for DNA amplification were compared, with three showing over-amplification of small circular ssDNA viruses, most notably GenomiPhi. Studies of viral particle stability in the presence of nuclease showed that most viral genomes were stable when protected in viral particles, but phage MS2 RNA was unexpectedly labile under some of the conditions tested. Comparison of Illumina 1000-cycle sequencing versus 300-cycle sequencing showed that longer reads supported generation of longer viral genome assemblies. Bacteriophage DNA can be modified by at least 12 different chemistries, raising the question of whether these modifications might block recovery in virome analytical protocols. We tested bacteriophage T4 DNA modified with glucosyl-hydroxymethylcytosine (ghmC) and hydroxymethylcytosine (hmC), and found that both were readily detected, though the recovery of ghmC-modified DNA was reduced. These studies together with published data help provide guidance for virome researchers optimizing analytical protocols.

microbiology↗

bioRxiv2025

Carbohydrate consumption drives adaptive mutations in Escherichia coli associated with increased risk for systemic infection

Dissemination of organisms from the gut microbiota is a major contributor to sepsis and critical illness. Patients with cirrhosis are prone to systemic infections and are commonly prescribed the carbohydrate lactulose to manage hepatic encephalopathy (HE) 1. Commensal metabolism of lactulose is believed to reduce pathobiont colonization through short-chain fatty acid production, but its direct effects on gut pathobionts remain unexplored 2. Here, we show that lactulose consumption unexpectedly selects for mutations in Escherichia coli lactose (lac) operon regulation, enhancing its metabolic fitness and colonization capacity. This is mediated by selection for constitutive expression of the lac operon through mutations in its regulatory components. Using in vitro systems, murine models, and clinical samples, we demonstrate that these mutations enable E. coli to exploit lactulose as a carbon source, bypassing host carbohydrate metabolism and increasing its intestinal colonization. Despite its long-standing use in HE treatment, we find that lactulose has a paradoxical association with risk of infection hospitalization in patients with cirrhosis in a large epidemiologic study. The emergence of lactulose-adapted E. coli strains could be suppressed by a dietary oligosaccharide that competitively inhibits lactulose uptake. These findings reveal a mechanism by which dietary substrates exert selective pressure on the microbiome, with implications for diet-based strategies to modulate microbial evolution and infection risk.

microbiology↗

bioRxiv2023

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.

microbiology↗

bioRxiv2023

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

BackgroundLinezolid 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). ObjectivesThe goals of this study were to determine the incidence of linezolid resistance in CF and identify molecular mechanisms for linezolid resistance. MethodsWe 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 ResultsBetween 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. ConclusionsLinezolid 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 PointLinezolid resistance arises through multiple genetic mechanisms and could be facilitated by mutator phenotypes. Linezolid resistance was transient, possibly due to growth disadvantage.

microbiology↗

bioRxiv2022

Efficient Pangenome Construction through Alignment-Free Residue Pangenome Analysis (ARPA)

Protein sequences can be transformed into vectors composed of counts for each amino acid (vector of Residue Counts; vRC) that are mathematically tractable and retain information about homology. We use vRCs to perform alignment-free, residue-based, pangenome analysis (ARPA; https://github.com/Arnavlal/ARPA). ARPA is 70-90 times faster at identifying homologous gene clusters compared to standard techniques, and offers rapid calculation, visualization, and novel phylogenetic approaches for pangenomes.

bioinformatics↗

bioRxiv2021

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.

genomics↗

bioRxiv2020

Emerging SARS-CoV-2 diversity revealed by rapid whole genome sequence typing.

BackgroundDiscrete classification of SARS-CoV-2 viral genotypes can identify emerging strains and detect geographic spread, viral diversity, and transmission events. MethodsWe developed a tool (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 each of 69,686 SARS-CoV-2 complete, high-quality genomes available from GISAID as of October 20th 2020. STs were then 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. ResultsGNUVID is a scalable tool for viral genotype classification (available at https://github.com/ahmedmagds/GNUVID) that can be used to quickly process tens of thousands of genomes. Our genotyping ST/CC analysis uncovered dynamic local changes in ST/CC prevalence and diversity with multiple replacement events in different states. We detected an average of 20.6 putative introductions and 7.5 exportations for each state. Effective viral diversity dropped in all states as shelter-in-place travel-restrictions went into effect and increased as restrictions were lifted. Interestingly, our analysis showed correlation between effective diversity and the date that state-wide mask mandates were imposed. ConclusionsOur classification tool uncovered multiple introduction and exportation events, as well as waves of expansion and replacement of SARS-CoV-2 genotypes in different states. Combined with future genomic sampling the GNUVID system could be used to track circulating viral diversity and identify emerging clones and hotspots.

genomics↗

bioRxiv2020

Structure-guided microbial targeting of antistaphylococcal prodrugs

Carboxy ester prodrugs have been widely employed as a means to increase oral absorption and potency of phosphonate antibiotics. Prodrugging can successfully mask problematic chemical features that prevent cellular uptake and can be used to target delivery of compounds to specific tissues. However, many carboxy ester promoieties are rapidly hydrolyzed by serum esterases, curbing their potential therapeutic applications. While carboxy ester-based prodrug targeting is feasible, it has seen limited use in microbes due to a paucity of information about the selectivity of microbial esterases. Here we identify the bacterial esterases, GloB and FrmB, that are required for carboxy ester prodrug activation 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, which revealed several promoieties likely to be serum esterase-resistant while still being microbially labile. These studies lay the groundwork for structure-guided design of anti-staphyloccal promoieties and expand the range of molecules to target staphyloccal pathogens.

biochemistry↗

bioRxiv2020

Rapid whole genome sequence typing reveals multiple waves of SARS-CoV-2 spread

As the pandemic SARS-CoV-2 virus has spread globally its genome has diversified to an extent that distinct clones can now be recognized, tracked, and traced. Identifying clonal groups allows for assessment of geographic spread, transmission events, and identification of new or emerging strains that may be more virulent or more transmissible. Here we present a rapid, whole genome, allele-based method (GNUVID) for assigning sequence types to sequenced isolates of SARS-CoV-2 sequences. This sequence typing scheme can be updated with new genomic information extremely rapidly, making our technique continually adaptable as databases grow. We show that our method is consistent with phylogeny and recovers waves of expansion and replacement of sequence types/clonal complexes in different geographical locations. GNUVID is available as a command line application (https://github.com/ahmedmagds/GNUVID).

genomics↗