Biology subjects

Putonti, C.

Publications and source records attributed to Putonti, C..

9 recordsLinked to original sources

bioRxiv2026

Selecting for lysis in a temperate phage population

Temperate phages represent an abundant source of phage diversity, but their capacity for lysogeny limits their therapeutic potential. Here, we asked whether repeated selection for lytic replication could drive a temperate phage toward an obligately lytic lifestyle. We serially propagated a cocktail of three temperate coliphages using a modified Appelmans protocol in which phage populations were independently passaged on eight E. coli hosts. Whole-genome sequencing revealed substantial changes in population composition and mutation frequencies. The Uetakevirus phage 1362 was undetectable after the first round, whereas the closely related P2-like phages in the cocktail dominated the evolving populations. Mutations repeatedly accumulated in lysis- and lysogeny-associated genes. Longitudinal genomic analysis revealed rapid fixation of mutations in the integrase coding region, while variants in lysA, lysB, and holin increased in frequency over successive rounds of selection. Protein structure modeling localized selected mutations to regions with potential functional significance. These include the DNA-binding domain of integrase and a surface-exposed region of LysA. Together, these results show that sustained selection for lytic replication can drive rapid and repeatable genomic adaptation in temperate phage populations.

evolutionary biology↗

bioRxiv2023

Cataloging the Phylogenetic Diversity of Human Bladder Bacterial Isolates

Although the human bladder is reported to harbor unique microbiota, our understanding of how these microbial communities interact with their human hosts is limited, mostly owing to the lack of isolates to test mechanistic hypotheses. Niche-specific bacterial collections and associated reference genome databases have been instrumental in expanding knowledge of the microbiota of other anatomical sites, e.g., the gut and oral cavity. To facilitate genomic, functional, and experimental analyses of the human bladder microbiota, here we present a bladder-specific bacterial reference collection comprised of 1134 genomes. These genomes were culled from bacterial isolates obtained by a metaculturomic method from bladder urine collected by transurethral catheterization. This bladder-specific bacterial reference collection includes 196 different species, including representatives of major aerobes and facultative anaerobes, as well as some anaerobes. It captures 72.2 % of the genera found when we reexamined previously published 16S rRNA gene sequencing of 392 adult female bladder urine samples. Comparative genomic analysis found that the taxonomies and functions of the bladder microbiota shared more similarities with the vaginal microbiota than the gut microbiota. Whole-genome phylogenetic and functional analyses of 186 bladder E. coli isolates and 387 gut E. coli isolates supports the hypothesis that phylogroup distribution and functions of E. coli strains differ dramatically between these two very different niches. This bladder-specific bacterial reference collection is a unique resource that will enable hypothesis-driven bladder microbiota research and comparison to isolates from other anatomical sites.

microbiology↗

bioRxiv2022

Urinary F plasmids reduce permissivity to coliphage infection

The microbial community of the urinary tract (urinary microbiota or urobiota) has been associated with human health. Bacteriophages (phages) and plasmids present in the urinary tract, like in other niches, may shape urinary bacteria dynamics. While urinary E. coli, often associated with urinary tract infection (UTI), and their phages have been catalogued for the urobiome, the dynamics of their interactions have yet to be explored. In this study, we characterized urinary E. coli F plasmids and their ability to decrease permissivity to E. coli phage (coliphage) infection. Putative F plasmids were present in 47 of 67 urinary E. coli isolates, and most of these plasmids carry genes that encode for toxin-antitoxin (TA) modules, antibiotic resistance, and/or virulence. Two urinary E. coli F plasmids, from urinary microbiota (UMB) 0928 and 1284, were conjugated into E. coli K-12 strains; plasmids included genes for antibiotic resistance and virulence. These plasmids, pU0928 and pU1284, decreased permissivity to coliphage infection by the laboratory phage P1vir and the urinary phages Greed and Lust. Furthermore, pU0928 could be maintained in E. coli K-12 for up to ten days in the absence of antibiotic resistance selection; this included maintenance of the antibiotic resistance phenotype and decreased permissivity to phage. Finally, we discuss how F plasmids present in urinary E. coli could play a role in coliphage dynamics and maintenance of antibiotic resistance in urinary E. coli. ImportanceThe microbial community of the urinary tract (urinary microbiota or urobiota) has been associated with human health. Bacteriophages (phages) and plasmids present in the urinary tract, like in other niches, may shape urinary bacteria dynamics. While urinary E. coli, often associated with urinary tract infection, and their phages have been catalogued for the urobiome, the dynamics of their interactions have yet to be explored. In this study, we characterized urinary E. coli F plasmids and their ability to decrease permissivity to E. coli phage (coliphage) infection. Two urinary E. coli F plasmids, each encoding antibiotic resistance and transferred by conjugation into naive laboratory E. coli K-12 strains decreased permissivity to coliphage infection. We propose a model by which F plasmids present in urinary E. coli could help to maintain antibiotic resistance of urinary E. coli.

microbiology↗

bioRxiv2022

Profiling the plasmid conjugation potential of urinary E. coli

Escherichia coli is often associated with urinary tract infection (UTI). Antibiotic resistance in E. coli is an ongoing challenge in managing UTI. Extrachromosomal elements - plasmids - are vectors for clinically relevant traits, such as antibiotic resistance, with conjugation being one of the main methods for horizontal propagation of plasmids in bacterial populations. Targeting of conjugation components has been proposed as a strategy to curb spread of plasmid-borne antibiotic resistance. Understanding the types of conjugative systems present in urinary E. coli isolates is fundamental to assessing the viability of these strategies. In this study, we profile the two well studied conjugation systems (F-type and P-type) in the draft genomes of 65 urinary isolates of E. coli obtained from bladder urine of adult women with and without UTI-like symptoms. Most of these isolates contained plasmids and we found that conjugation genes are abundant/ubiquitous, diverse, and often associated with IncF plasmids. To validate conjugation of these urinary plasmids, we conjugated the plasmids from two urinary isolates, UMB1223 (predicted to have F-type genes) and UMB1284 (predicted to have P-type genes), into the K-12 E. coli strain MG1655. Overall, the findings of this study support the notion that care should be taken in targeting any individual component of a urinary E. coli isolates conjugation system, given the inherent mechanistic redundancy, gene diversity, and different types of conjugation systems in this population.

microbiology↗

bioRxiv2021

Phylogenomic study of Staphylococcus aureus and Staphylococcus haemolyticus clinical isolates from Egypt

Antibiotic resistant Staphylococcus infections are a global concern, with increasing cases of resistant Staphylococcus aureus and Staphylococcus haemolyticus found circulating in the Middle East. While extensive surveys have described the prevalence of resistant infections in Europe, Asia, and North America, the population structure of resistant staphylococcal Middle Eastern clinical isolates is poorly characterized. We performed whole genome sequencing of 56 S. aureus and 10 S. haemolyticus isolates from Alexandria Main University Hospital. Supplemented with additional publicly available genomes from the region (34 S. aureus and 6 S. haemolyticus), we present the largest genomic study of staphylococcal Middle Eastern isolates. These genomes include 20 S. aureus multilocus sequence typing (MLST) types and 9 S. haemolyticus MLSTs, including 3 and 1 new MLSTs, respectively. Phylogenomic analyses of each species core genome largely mirrored MLSTs, irrespective of geographical origin. The hospital-acquired spa t037/SCCmec III/MLST CC8 clone represented the largest clade, comprising 22% of S. aureus isolates. Similar to other regional genome surveys of S. aureus, the Middle Eastern isolates have an open pangenome, a strong indicator of gene exchange of virulence factors and antibiotic resistance genes with other reservoirs. We recommend stricter implementation of antibiotic stewardship and infection control plans in the region. Impact StatementStaphylococci are under-studied despite their prevalence within the Middle East. Methicillin-resistant Staphylococcus aureus (MRSA) is endemic to hospitals in this region, as are other antibiotic-resistant strains of S. aureus and S. haemolyticus. To provide insight into the strains currently in circulation within Egypt, we performed whole genome sequencing of 56 S. aureus and 10 S. haemolyticus isolates from Alexandria Main University Hospital (AMUH). Through analysis of these genomes, as well as other genomes of isolates from the Middle East, we were able to produce a more complete picture of the current diversity than traditional molecular typing strategies. Furthermore, the S. haemolyticus genome analyses provide the first insight into strains found in Egypt. Our analysis of resistance and virulence mechanisms carried by these strains provides invaluable insight into future plans of antibiotic stewardship and infection control within the region. Data SummaryRaw sequencing reads and assembled genomes can be found at BioProject Accession number PRJNA648411 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA648411).

microbiology↗

bioRxiv2021

Rephine.r: a pipeline for correcting gene calls and clusters to improve phage pangenomes and phylogenies

BackgroundA pangenome is the collection of all genes found in a set of related genomes. For microbes, these genomes are often different strains of the same species, and the pangenome offers a means to compare gene content variation with differences in phenotypes, ecology, and phylogenetic relatedness. Though most frequently applied to bacteria, there is growing interest in adapting pangenome analysis to bacteriophages. However, working with phage genomes presents new challenges. First, most phage families are under-sampled, and homologous genes in related viruses can be difficult to identify. Second, homing endonucleases and intron-like sequences may be present, resulting in fragmented gene calls. Each of these issues can reduce the accuracy of standard pangenome analysis tools. MethodsWe developed an R pipeline called Rephine.r that takes as input the gene clusters produced by an initial pangenomics workflow. Rephine.r then proceeds in two primary steps. First, it identifies three common causes of fragmented gene calls: 1) indels creating early stop codons and new start codons; 2) interruption by a selfish genetic element; and 3) splitting at the ends of the reported genome. Fragmented genes are then fused to create new sequence alignments. In tandem, Rephine.r searches for distant homologs separated into different gene families using Hidden Markov Models. Significant hits are used to merge families into larger clusters. A final round of fragment identification is then run, and results may be used to infer single-copy core genomes and phylogenetic trees. ResultsWe applied Rephine.r to three well-studied phage groups: the Tevenvirinae (e.g. T4), the Studiervirinae (e.g. T7), and the Pbunaviruses (e.g. PB1). In each case, Rephine.r recovered additional members of the single-copy core genome and increased the overall bootstrap support of the phylogeny. The Rephine.r pipeline is provided through GitHub (https://www.github.com/coevoeco/Rephine.r) as a single script for automated analysis and with utility functions and a walkthrough for researchers with specific use cases for each type of correction.

bioinformatics↗

bioRxiv2019

UP{varphi} phages, a new group of filamentous phages found in several members of Enterobacteriales

Filamentous phages establish chronic infections in their bacterial hosts, and new phages are secreted by infected bacteria for multiple generations, typically without causing host death. Often, these viruses integrate in their hosts genome by co-opting the hosts XerCD recombinase system. In several cases, these viruses also encode genes that increase bacterial virulence in plants and animals. Here, we describe a new filamentous phage, UP{phi}901, which we originally found integrated in a clinical isolate of uropathogenic Escherichia coli. UP{phi}901 and closely related phages can be found in published genomes of over 200 other bacteria, including strains of Citrobacter koseri, Salmonella enterica, Yersinia enterocolitica, and Klebsiella pneumoniae. Its closest relatives are consistently found in urine or in the blood and feces of patients with urinary tract infections. More distant relatives can be found in isolates from other environments, including sewage, water, soil, and contaminated food. Each of these phages, which we collectively call "UP{phi} viruses," also harbors two novel genes of unknown function.

microbiology↗

bioRxiv2017

Gene networks provide a high-resolution view of bacteriophage ecology

Bacteriophages are the most abundant and diverse biological entities on the planet, and new phage genomes are being discovered at a rapid pace from metagenomes. As more novel, uncultured phage genomes are published, new tools are needed for placing these genomes in an ecological and evolutionary context. Phages are difficult to study with phylogenetic methods, because they exchange genes regularly, and no single gene is conserved across all phages. Instead, genome-level networks have been used to group similar viruses into clusters for taxonomy. Here, we show that gene-level networks provide a high-resolution view of phage genetic diversity and offer a novel perspective on virus ecology. To that end, we developed a method that identifies informative associations between a phages annotated host and clusters of genes in the network. Given these associations, we were able to predict a phages host with 86% accuracy at the genus level, while also identifying genes that underlie these virus-host interactions. This approach, thus, provides one of the most accurate means of host prediction while also pointing to directions for future empirical work.

microbiology↗

bioRxiv2017

Exploring the Diversity of Bacillus whole genome sequencing projects using Peasant, the Prokaryotic Assembly and Annotation Tool

BackgroundThe persistent decrease in cost and difficulty of whole genome sequencing of microbial organisms has led to a dramatic increase in the number of species and strains characterized from a wide variety of environments. Microbial genome sequencing can now be conducted by small laboratories and as part of undergraduate curriculum. While sequencing is routine in microbiology, assembly, annotation and downstream analyses still require computational resources and expertise, often necessitating familiarity with programming languages. To address this problem, we have created a light-weight, user-friendly tool for the assembly and annotation of microbial sequencing projects.\n\nResultsThe Prokaryotic Assembly and Annotation Tool, Peasant, automates the processes of read quality control, genome assembly, and annotation for microbial sequencing projects. High-quality assemblies and annotations can be generated by Peasant without the need of programming expertise or high-performance computing resources. Furthermore, statistics are calculated so that users can evaluate their sequencing project. To illustrate the computational speed and accuracy of Peasant, the SRA records of 322 Illumina platform whole genome sequencing assays for Bacillus species were retrieved from NCBI, assembled and annotated on a single desktop computer. From the assemblies and annotations produced, a comprehensive analysis of the diversity of over 200 high-quality samples was conducted, looking at both the 16S rRNA phylogenetic marker as well as the Bacillus core genome.\n\nConclusionsPeasant provides an intuitive solution for high-quality whole genome sequence assembly and annotation for users with limited programing experience and/or computational resources. The analysis of the Bacillus whole genome sequencing projects exemplifies the utility of this tool. Furthermore, the study conducted here provides insight into the diversity of the species, the largest such comparison conducted to date.

bioinformatics↗