Biology subjects

Azarian, T.

Publications and source records attributed to Azarian, T..

10 recordsLinked to original sources

bioRxiv2025

Genomic and Phenotypic Characterization of Mupirocin Resistant Staphylococcus aureus Clinical Isolates

BackgroundColonization with Staphylococcus aureus is a risk factor for subsequent infection. Decolonization with the topical antibiotic mupirocin is effective and reduces the risk of subsequent S. aureus infection for both methicillin-sensitive (MSSA) and methicillin-resistant (MRSA) strains but may select for mupirocin-resistant isolates. MethodsWe characterized oxacillin and mupirocin susceptibility amongst 384 S. aureus strains isolated from clinical samples isolated 2017-2023 in Tampa, Florida, spanning strains collected before and after the onset of the COVID-19 pandemic. Whole genome sequencing of bacterial isolates was conducted in parallel and correlated with drug susceptibility profiles. ResultsMupirocin resistance (MupR) was nearly exclusively present in MRSA strains (103/106 97.1% of MupR; 103/299 34.4% of MRSA). Although our hospital protocol for decolonization shifted to povidone iodine in the Post-COVID period, the overall prevalence of MupR did not change in Pre-COVID and Post-COVID samples (28.9% vs 26%). Genotype correlated with antibiotic susceptibility with low level MupR (MupLR), linked to mutations in ileS and high level MupR (MupHR), linked to the presence of mupA. Genome analysis revealed that most MupR strains fell into three sequence types (ST) falling into two major clonal complexes (CC): CC8 ST8 (including Community-Associated MRSA strains USA300 and USA500), CC5 ST5 (associated with Healthcare-Associated MRSA such as USA100), and CC5 ST3390. ST3390 isolates had the highest prevalence of MupR (30/36 83%; MupHR 20/36 55.6%; MupLR 10/36 27.8%). ConclusionsMupirocin resistance was prevalent in our hospital MRSA strains. We also found evidence for emergence and persistence of ST3390 MRSA-MupR strains in Florida. Key pointsO_LIIn a survey of clinical isolates in Florida, 34.4% of MRSA strains were mupirocin resistant. C_LIO_LIMupirocin resistance correlated with mutations in ileS or carriage of mupA. C_LIO_LIWe found evidence for emergence of MRSA mupirocin-resistant strains that were sequence type ST3390. C_LI

microbiology↗

bioRxiv2025

Emergence of ST3390: A non-pigmented HA-MRSA clone with enhanced virulence

BackgroundOne of the most successful and widely-distributed hospital-associated lineages of MRSA is CC5. These strains are known from widespread antibiotic resistance, but less severe disease than CA-MRSA counterparts. Recently, CC5 descendant lineages have appeared globally with hypervirulent properties. MethodsHerein we use genomic analyses to study the epidemiology of a rare CC5 MRSA sequence type, ST3390, circulating within Tampa General Hospital (TGH). We employ genetic tools alongside in vitro and in vivo models of virulence to study the pathogenic capabilities of strains. ResultsTo date, there have only been 50 recorded instances of infection caused by ST3390 globally, with 36 of those occurring at TGH. Genomic analysis of strains identified numerous spa-types, with a t010 cluster found only at TGH. Exploration of AMR genes detected the presence of unique hybrid SCCmec types, with [~]90% of TGH strains possessing components of SCCmecIa, SCCmecIIa and/or SCCmecVIII. Phenotypically, all ST3390 strains lack the staphyloxanthin pigment, which is mediated by a conserved 6 aa in frame deletion within the staphyloxanthin biosynthesis protein CrtN. ST3390 strains display high levels of cytotoxicity towards human neutrophils compared to other CC5 lineages, with several isolates displaying hypervirulence in animal models of infection. ConclusionsThis is the first study to characterize the pathogenicity and genomic architecture of the rare MRSA lineage ST3390. Our work provides a deeper understanding of the clonal expansion of CC5, and the wider diversification of S. aureus isolates within patient populations.

microbiology↗

bioRxiv2024

Linkage-based ortholog refinement in bacterial pangenomes with CLARC

Bacterial genomes exhibit significant variation in gene content and sequence identity. Pangenome analyses explore this diversity by classifying genes into core and accessory clusters of orthologous groups (COGs). However, strict sequence identity cutoffs can misclassify divergent alleles as different genes, inflating accessory gene counts. CLARC (Connected Linkage and Alignment Redefinition of COGs) [https://github.com/IndraGonz/CLARC] improves pangenome analyses by condensing accessory COGs using functional annotation and linkage information. Through this approach, orthologous groups are consolidated into more practical units of selection. Analyzing 8,000+ Streptococcus pneumoniae genomes, CLARC reduced accessory gene estimates by more than 30% and improved evolutionary predictions based on accessory gene frequencies. By refining COG definitions, CLARC offers critical insights into bacterial evolution, aiding genetic studies across diverse populations.

bioinformatics↗

bioRxiv2024

Variation in transformation frequency and competence gene expression among serotype 3 Streptococcus pneumoniae

BackgroundStreptococcus pneumoniae is a human commensal and the causative agent of pneumococcal disease. Pneumococci are naturally competent - able to uptake exogenous DNA from the environment and incorporate it into their genome through homologous and non- homologous recombination. Recombination has significantly shaped the evolutionary history of S. pneumoniae, as it allows pneumococci to rapidly adapt. Recombination frequencies vary considerably across pneumococcal populations, yet underlying mechanisms for these variations are not well understood. Entry and exit into competence, a state in which the cell can uptake DNA, is tightly regulated through transcriptional changes. MethodsTo elucidate differences in transformation frequency among strains as well as the underlying genetic mechanisms, we carried out in-vitro competence assays and measured gene expression changes during the competent state using RNA sequencing of representative strains belonging to Serotype 3 clonal complex (CC) 180 and a non-CC180 comparison. ResultsWe observed consistent differences in transformation frequencies among groups, which correlated with variation in differentially expressed genes during competence. While all strains exhibited a similar response to competence stimulating peptide (CSP) for early competence genes, we observed variation in expression of late competence genes, which encode the DNA uptake apparatus, DNA repair and recombination proteins. We also observed differences in expression of genes linked to bacteriocin production, which may partially explain observed population-level differences. Further genomic analysis suggests variation in promoter sequences governing late competence genes may be slowing transition from early to late components of the competence pathway. ConclusionsWe show that there is considerable variation in competence even among closely related strains belonging to the same CC and that this variation may be the result of subtle genomic differences. Additional studies are needed to assess the phenotypic impact of genomic variations.

microbiology↗

bioRxiv2023

Improving bacterial metagenomic research through long read sequencing

Metagenomic sequencing analysis is central to investigating microbial communities in clinical and environmental studies. Short read sequencing remains the primary data type for metagenomic research, however, long read sequencing promises advantages of improved metagenomic assembly and resolved taxonomic identification. To assess the comparative performance of short and long read sequencing data for metagenomic analysis, we simulated short and long read datasets using increasingly complex metagenomes comprised of 10, 20, and 50 microbial taxa. In addition, an empirical dataset of paired short and long read data from mouse fecal pellets was generated to assess feasibility. We compared metagenomic assembly quality, taxonomic classification capabilities, and metagenome-assembled genome recovery rates for both simulated and real metagenomic sequence data. We show that long read sequencing data significantly improves taxonomic classification capabilities and assembly quality. For simulated long read datasets, metagenomic assemblies were completer and more contiguous with higher rates of metagenome-assembled genome recovery. This resulted in more precise taxonomic classifications. Analysis of empirical data demonstrated that sequencing technology directly affects compositional results. Overall, we highlight strengths of long read sequencing for metagenomic studies of microbial communities over traditional short read approaches. Long read sequencing improved the accuracy of classification and abundance estimation. These results will aid researchers when considering which sequencing platforms to use for metagenomic projects. Data descriptionThe experimental metagenomic sequence data used for comparison of short and long read data from the same source are available from NCBIs Sequence Read Archive (SRA) under Bioproject accession ID PRJNA1092431.

bioinformatics↗

bioRxiv2022

A collaborative approach to improving representation in viral genomic surveillance

The lack of routine viral genomic surveillance delayed the initial detection of SARS-CoV-2, allowing the virus to spread unfettered at the outset of the U.S. epidemic. Over subsequent months, poor surveillance enabled variants to emerge unnoticed. Against this backdrop, long-standing social and racial inequities have contributed to a greater burden of cases and deaths among minority groups. To begin to address these problems, we developed a new variant surveillance model geared toward building microbial genome sequencing capacity at universities in or near rural areas and engaging the participation of their local communities. The resulting genomic surveillance network has generated more than 1,000 SARS-CoV-2 genomes to date, including the first confirmed case in northeast Louisiana of Omicron, and the first and sixth confirmed cases in Georgia of the emergent BA.2.75 and BQ.1.1 variants, respectively. In agreement with other studies, significantly higher viral gene copy numbers were observed in Delta variant samples compared to those from Omicron BA.1 variant infections, and lower copy numbers were seen in asymptomatic infections relative to symptomatic ones. Collectively, the results and outcomes from our collaborative work demonstrate that establishing genomic surveillance capacity at smaller academic institutions in rural areas and fostering relationships between academic teams and local health clinics represent a robust pathway to improve pandemic readiness. Author summaryGenomic surveillance involves decoding a pathogens genetic code to track its spread and evolution. During the pandemic, genomic surveillance programs around the world provided valuable data to scientists, doctors, and public health officials. Knowing the complete SARS-CoV-2 genome has helped detect the emergence of new variants, including ones that are more transmissible or cause more severe disease, and has supported the development of diagnostics, vaccines, and therapeutics. The impact of genomic surveillance on public health depends on representative sampling that accurately reflects the diversity and distribution of populations, as well as rapid turnaround time from sampling to data sharing. After a slow start, SARS-CoV-2 genomic surveillance in the United States grew exponentially. Despite this, many rural regions and ethnic minorities remain poorly represented, leaving significant gaps in the data that informs public health responses. To address this problem, we formed a network of universities and clinics in Louisiana, Georgia, and Mississippi with the goal of increasing SARS-CoV-2 sequencing volume, representation, and equity. Our results demonstrate the advantages of rapidly sequencing pathogens in the same communities where the cases occur and present a model that leverages existing academic and clinical infrastructure for a powerful decentralized genomic surveillance system.

microbiology↗

bioRxiv2019

Long-term intrahost evolution of methicillin-resistant Staphylococcus aureus among cystic fibrosis patients with respiratory carriage

Staphylococcus aureus is the most commonly identified airway colonizer of cystic fibrosis (CF) patients, and infections with methicillin-resistant S. aureus (MRSA) are associated with poor outcomes. Yet, little is known about the intrahost evolution of S. aureus among CF patients. We investigated convergent evolution and adaptation of MRSA among four CF patients with long-term respiratory carriage. For each patient, we performed whole-genome sequencing on an average of 21 isolates (range: 19-23) carried for a mean of 1,403 days (range: 903-1,679), including 25 pairs of isolates collected on the same day. We then assessed intrahost diversity, population structure, evolutionary history, and signatures of adaptation in the context of patient age, antibiotic treatment, and co-colonizing microbes. We conducted tests of gene-wide diversifying selection and identified instances of switched intergenic regions (IGRs), which may be associated with gene expression. Phylogenetic analysis delineated distinct multilocus sequence type ST5 (n=3) and ST72 (n=1) clonal populations in addition to sporadic, non-clonal isolates, and uncovered a putative transmission event. Variation in antibiotic resistance was observed within clonal populations, even among isolates collected on the same day. Coalescent analysis revealed rates of molecular evolution ranging from 2.21 to 8.64 nucleotide polymorphisms per genome per year. Estimated lineage ages were consistent with acquisition of colonization in early childhood and subsequent persistence of multiple sub-populations. Selection analysis focused on 1,622 core genes shared by all four clonal populations (n=79). Eleven genes were variable in three subjects - most notable, ATP-dependent protease clpX, 2-oxoglutarate dehydrogenase odhA, fmtC, and transcription-repair coupling factor mfd. Only one gene, staphylococcal protein A (spa), was found to have evidence of gene-wide diversifying selection. We identified three instances of intrahost IGR switching events, two of which flanked genes related to quorum sensing. The ecology of microbes in the airways of CF patients is undeniably complex, posing challenges for management. We illustrate appreciable intrahost diversity as well as persistence of a dominant lineage. We also show that intrahost adaptation is a continual process, despite purifying selective pressure, and provide targets that should be investigated further for their function in CF adaptation.\n\nContribution to the FieldStaphylococcus aureus is the most commonly identified airway colonizer of cystic fibrosis (CF) patients, and infections with methicillin-resistant S. aureus (MRSA) are associated with poor outcomes. Yet, little is known about the intrahost evolution of S. aureus among CF patients. We investigated convergent evolution and adaptation of MRSA among four CF patients with long-term respiratory carriage. We found that each patient possessed a single predominant strain in addition to transient non-clonal isolates. Considerable genomic diversity was observed among intrahost populations including variation in antibiotic resistance, even among isolates collected on the same day. This finding has implications for treatment. Estimated lineage ages were consistent with acquisition of colonization in early childhood and subsequent persistence of multiple sub-populations. The scarcity of non-synonymous substitutions suggests chronic S. aureus carriage in CF patients provides a sufficient opportunity for purifying selection to act. Eleven genes were found to be variable in three patients and present possible candidates for loci experiencing gene-wide diversifying selection. Overall, we show that intrahost adaptation is a continual process, despite purifying selective pressure, and provide targets that should be investigated further for their function in CF adaptation.

evolutionary biology↗

bioRxiv2018

Prediction of post-vaccine population structure of Streptococcus pneumoniae using accessory gene frequencies

Predicting how pathogen populations will change over time is challenging. Such has been the case with Streptococcus pneumoniae, an important human pathogen, and the pneumococcal conjugate vaccines (PCVs), which target only a fraction of the strains in the population. Here, we use the frequencies of accessory genes to predict changes in the pneumococcal population after vaccination, hypothesizing that these frequencies reflect negative frequency-dependent selection (NFDS) on the gene products. We find that the standardized predicted fitness of a strain estimated by an NFDS-based model at the time the vaccine is introduced enables to predict whether the strain increases or decreases in prevalence following vaccination. Further, we are able to forecast the equilibrium post-vaccine population composition and assess the invasion capacity of emerging lineages. Overall, we provide a method for predicting the impact of an intervention on pneumococcal populations with potential application to other bacterial pathogens in which NFDS is a driving force.

evolutionary biology↗

bioRxiv2018

Global emergence and population dynamics of divergent serotype 3 CC180 pneumococci

Streptococcus pneumoniae serotype 3 remains a significant cause of morbidity and mortality worldwide, despite inclusion in the 13-valent pneumococcal conjugate vaccine (PCV13). Serotype 3 increased in carriage since the implementation of PCV13 in the United States, while invasive disease rates remain unchanged. We investigated the persistence of serotype 3 in carriage and disease, through genomic analyses of a global sample of 301 serotype 3 isolates of the Netherlands3-31 (PMEN31) clone CC180, combined with associated patient data and PCV utilization among countries of isolate collection. We assessed phenotypic variation between dominant clades in capsule charge (zeta potential), capsular polysaccharide shedding, and susceptibility to opsonophagocytic killing, which have previously been associated with carriage duration, invasiveness, and vaccine escape. We identify a recent shift in the CC180 population attributed to a lineage termed Clade II, which was estimated by Bayesian coalescent analysis to have first appeared in 1968 [95% HPD: 1939-1989] and increased in prevalence and effective population size thereafter. Clade II isolates are divergent from the pre-PCV13 serotype 3 population in non-capsular antigenic composition, competence, and antibiotic susceptibility, the last resulting from the acquisition of a Tn916-like conjugative transposon. Differences in recombination rates among clades correlated with variations in the ATP-binding subunit of Clp protease as well as amino acid substitutions in the comCDE operon. Opsonophagocytic killing assays elucidated the low observed efficacy of PCV13 against serotype 3. Variation in PCV13 use among sampled countries was not independently correlated with the CC180 population shift; therefore, genotypic and phenotypic differences in protein antigens and, in particular, antibiotic resistance may have contributed to the increase of Clade II. Our analysis emphasizes the need for routine, representative sampling of isolates from disperse geographic regions, including historically under-sampled areas. We also highlight the value of genomics in resolving antigenic and epidemiological variations within a serotype, which may have implications for future vaccine development.\n\nAuthor SummaryStreptococcus pneumoniae is a leading cause of bacterial pneumoniae, meningitis, and otitis media. Despite inclusion in the most recent pneumococcal conjugate vaccine, PCV13, serotype 3 remains epidemiologically important globally. We investigated the persistence of serotype 3 using whole-genome sequencing data form 301 isolates collected among 24 countries from 1993-2014. Through phylogenetic analysis, we identified three distinct lineages within a single clonal complex, CC180, and found one has recently emerged and grown in prevalence. We then compared genomic difference among lineages as well as variations in pneumococcal vaccine use among sampled countries. We found that the recently emerged lineage, termed Clade II, has a higher prevalence of antibiotic resistance compared to other lineages, diverse surface protein antigens, and a higher rate of recombination, a process by which bacteria can uptake and incorporate genetic material from its surroundings. Differences in vaccine use among sampled countries did not appear to be associated with the emergence of Clade II. We highlight the need to routine, representative sampling of bacterial isolates from diverse geographic areas and show the utility of genomic data in resolving epidemiological differences within a pathogen population.

evolutionary biology↗

bioRxiv2017

Population genomics of pneumococcal carriage in Massachusetts children following PCV-13 introduction

BackgroundThe 13-valent pneumococcal conjugate vaccine (PCV-13) was introduced in the United States in 2010. Using a large pediatric carriage sample collected from shortly after the introduction of PCV-7 to several years after the introduction of PCV-13, we investigate alterations in the composition of the pneumococcal population following the introduction of PCV-13, evaluating the extent to which the post-vaccination non-vaccine type (NVT) population mirrors that from prior to vaccine introduction and the effect of PCV-13 on vaccine type lineages.\n\nMethods and FindingsDraft genome assemblies from 736 newly sequenced and 616 previously published pneumococcal carriages isolates from children in Massachusetts between 2001 and 2014 were analyzed. Isolates were classified into one of 22 sequence clusters (SCs) on the basis of their core genome sequence. We calculated the SC diversity for each sampling period as the probability that any two randomly drawn isolates from that period belong to different SCs. The sampling period immediately after the introduction of PCV-13 (2011) was found to have higher diversity than preceding (2007) or subsequent (2014) sampling periods (Simpsons D 2007: 0.915 95% CI [0.901, 0.929]; 2011: 0.935 [0.927, 0.942]; 2014: 0.912 [0.901, 0.923]). Amongst NVT isolates, we found the distribution of SCs in 2011 to be significantly different from that in 2007 or 2014 (Fishers Exact Test p=0.018, 0.0078), but did not find a difference comparing 2007 to 2014 (Fishers Exact Test p=0.24), indicating greater similarity between samples separated by a longer time period than between samples from closer time periods. We also found changes in the accessory gene content of the NVT population between 2007 and 2011 to have been reduced by 2014. Amongst the new serotypes targeted by PCV-13, four were present in our sample. The proportion of our sample composed of PCV-13-only vaccine serotypes 19A, 6C, and 7F decreased between 2007 and 2014, but no such reduction was seen for serotype 3. We did, however, observe differences in the genetic composition of the pre- and post-PCV-13 serotype 3 population. Our isolates were collected during discrete sampling periods from a small geographic area, which may limit the generalizability our findings.\n\nConclusionPneumococcal diversity increased immediately following the introduction of PCV-13, but subsequently returned to pre-vaccination levels. This is reflected in the distribution of NVT lineages, and, to a lesser extent, their accessory gene frequencies. As such, there may be a period during which the population is particularly disrupted by vaccination before returning to a more stable distribution. The persistence and shifting genetic composition of serotype 3 is a concern and warrants further investigation.

evolutionary biology↗