Biology subjects

Gonzalez-Escalona, N.

Publications and source records attributed to Gonzalez-Escalona, N..

12 recordsLinked to original sources

bioRxiv2026

BoNTax: a curated genome-based framework for taxonomic classification and characterization of Clostridium botulinum Group I and Clostridium sporogenes

Clostridium botulinum Group I and Clostridium sporogenes are closely related genomic lineages for which historical toxin-based nomenclature can obscure species-level relationships. We developed BoNTax, a curated taxonomy-first framework that separates average nucleotide identity (ANI)-based species assignment from botulinum neurotoxin (BoNT) characterization while integrating assembly quality assessment, multilocus sequence typing (MLST), AMRFinderPlus-based detection of bont genes, other virulence-associated genes, and antimicrobial resistance genes into standardized outputs. Analysis of 966 genomes informed genome-size criteria and the selection of 46 representative references comprising 22 C. botulinum Group I and 24 C. sporogenes genomes. Pairwise ANI resolved these references into two distinct species clusters: within-species ANI values were >95%, whereas between-species values were <95%, supporting a 95% species-level assignment threshold. BoNTax was implemented in GalaxyTrakr as complementary FASTQ- and FASTA-input workflows that converge on a common downstream analysis. The workflow completed analysis of all 42 validation genomes. Among the 32 genomes with a reported taxonomic identity, 29 assignments were concordant and three prompted taxonomic re-evaluations. For the ten genomes reported as taxonomically unknown, BoNTax provided ANI-based assignments. Of the three discordances, BT-22100019 was assigned to C. sporogenes rather than the reported C. botulinum Group I, whereas R1125/03 and R1135/03 showed 99.96% ANI to C. caseinilyticum DSM 46279, supporting their identification as C. caseinilyticum. MLST assignments were concordant for genomes with established profiles except PNUSAB000061, for which ST183 was not assigned by the MLST database version used in this analysis. BoNT type and subtype detection was fully concordant across toxigenic genomes, including bivalent strains. Thirty-one genomes met the predefined assembly-size QC criterion, whereas 11 received QC warnings, including all eight datasets representing four known mixed cultures. BoNTax therefore provides a reproducible genomic framework for resolving closely related clostridial lineages, identifying taxonomic annotations requiring re-evaluation, and reporting clinically relevant toxin information without conflating toxin genotype with species identity.

genomics↗

bioRxiv2026

Genomic characterization of an atypical hydrogen sulfide-negative Salmonella enterica serovar Senftenberg strain lacking somatic antigen expression isolated from cooked mussels

Atypical Salmonella enterica strains that evade conventional detection pose significant challenges to food safety surveillance. A hydrogen sulfide (H2S)-negative and serologically untypable S. enterica strain (SF1060) was detected by qPCR from cooked farmed mussels in Galicia, Spain, and characterized using phenotypic and genomic approaches. Despite typical biochemical profiles, SF1060 failed to produce black colonies on XLD agar and lacked detectable somatic antigens by conventional serotyping. Hybrid genome assembly using Nanopore and Illumina sequencing yielded a closed chromosome and five plasmids. In silico analyses identified the strain as S. Senftenberg ST14. Comparative genomics revealed a chromosomal inversion at the rfb operon (encoding enzymes needed to synthesize deoxysugars and O antigens) mediated by IS5-family transposase ISEc68, which truncated the rfbD gene and separated the reminding rfb genes at rfbD, disrupting O-antigen biosynthesis, explaining the serotyping failure. The phs operon responsible for H2S production lacked premature stop codons, suggesting the H2S-negative phenotype results from an alternative mechanism. This study demonstrates how whole-genome sequencing resolves identification of atypical strains that fail culture-based detection and emphasizes the critical need for molecular surveillance methods in seafood safety programs, particularly in regions where atypical S. enterica variants may be endemic. Importance StatementPathogen surveillance is in a constant race against microbial evolution. The phenotypic methods used to detect and isolate foodborne bacteria like Salmonella enterica from foods are effective, but only if the pathogens retain their expected characteristics. This study provides a clear genetic snapshot of how Salmonella enterica can adapt to evade detection. This study characterizes a strain from a marine environment that had undergone genetic rearrangement, leading to the concurrent loss of a key biochemical marker and the somatic antigens essential for serological typing. Crucially, only genomic analysis could provide a definitive explanation for this serotyping failure; the underlying mechanism would have remained unknown using conventional methods alone. This work demonstrates that relying on a fixed set of phenotypic traits is an increasingly fragile strategy and highlights why genomic surveillance must become a routine component of food safety programs to keep pace with pathogen evolution.

microbiology↗

bioRxiv2025

centriflaken: an automated data analysis pipeline for assembly and in silico analyses of foodborne pathogens from metagenomic samples

Rapid and comprehensive analysis of metagenomic data from samples associated with foodborne outbreaks is of critical importance in food safety. Equally important is the need for automated analysis pipelines that allow the rapid and effective construction of metagenomic assembled genomes (MAGs) to enable bacterial source-tracking from metagenomic data. Here, we present centriflaken, an automated precision metagenomics pipeline for detecting and characterizing Shiga toxin-producing Escherichia coli (STEC) from metagenomic data. centriflaken streamlines the process of generating metagenome-assembled genomes (MAGs) and conducting in silico analyses of STECs, significantly reducing the time and manual effort required for comprehensive pathogen profiling. centriflaken was validated using Oxford Nanopore long-read sequencing data from agricultural water enrichments, successfully reproducing results from our previous study that involved multiple manual bioinformatics steps (Maguire et al., 2021). The tools efficacy was further demonstrated through its application to ZymoBIOMICS microbial community standards and 21 additional irrigation water samples, completing STEC precision metagenomics analyses in less than 7 hours per sample. centriflakens versatility allows for the analysis of user-defined taxa beyond STEC, including other foodborne pathogens like Listeria monocytogenes or Salmonella. The pipeline generates comprehensive summary plots and tables, accessible through a MultiQC HTML report. Designed for portability, centriflaken packages all software dependencies within containers and virtual environments. This open-source tool is available on GitHub under the MIT license (https://github.com/CFSAN-Biostatistics/centriflaken), offering a powerful resource for rapid, automated pathogen detection and characterization in food safety applications. Author summaryMetagenomic sequencing, particularly using nanopore technology, generates vast amounts of data that are challenging to analyze, especially when searching for specific pathogens like Shiga toxin-producing Escherichia coli (STEC). This challenge is compounded when processing multiple samples simultaneously. The pipeline "centriflaken" was developed to streamline this complex process, automating the extraction of E. coli reads from metagenomic data and performing in-silico characterization of potential STEC present in the samples. centriflaken builds on the concept of precision metagenomics, employing a suite of automated data analysis workflows powered by Nextflow. The pipeline processes metagenomic data, generates metagenome-assembled genomes (MAGs), and conducts in-silico analyses as described in Maguire et al. (2021). By running steps in parallel, centriflaken enables users without extensive bioinformatics skills or STEC genomic knowledge to efficiently analyze complex metagenomic data. Key features of centriflaken include: 1) Automated workflow for STEC detection and characterization, 2) Parallel processing for improved efficiency, 3) User-friendly interface for non-specialists, 4) Scalability for analyzing multiple samples simultaneously, and 5) Compatibility with high-performance computing (HPC) clusters and cloud environments. This freely available software democratizes complex metagenomic analysis, making it accessible to a broader range of researchers and food safety professionals.

bioinformatics↗

bioRxiv2024

Single laboratory evaluation of the (Q20+) nanopore sequencing kit for bacterial outbreak investigations

This study aimed to evaluate the potential of Oxford Nanopore Technologies (ONT) GridION with Q20+ chemistry as a rapid and accurate method for identifying and clustering foodborne pathogens. The study focuses on assessing whether ONT Q20+ technology could offer near real-time pathogen identification, including SNP differences, serotypes, and antimicrobial resistance genes, to overcome the drawbacks of existing methodologies. This pilot study evaluated different combinations of two DNA extraction methods (Maxwell RSC Cultured Cell DNA kit, and Monarch high molecular weight extraction kits) and two ONT library preparation protocols (ligation and the rapid barcoding sequencing kit) using five well-characterized strains representing diverse foodborne pathogens. The results showed that any combination of extraction and sequencing kits produced high-quality closed bacterial genomes. However, there were variations in assembly length and genome completeness based on different combinations of methods, indicating the need for further optimization. in silico analyses demonstrated that the Q20+ nanopore sequencing chemistry accurately identified species, genotyped, and detected virulence factors comparable to Illumina sequencing. Phylogenomic clustering methods showed that ONT assemblies clustered with reference genomes, although some indels and SNP differences were observed. There were also differences on SNP accuracy among the different species. The observed SNP differences were likely due to sequencing and analysis processes rather than genetic variations in the sampled bacteria. The study also compared a change in the basecaller model with the previous model (SUP 4Khz 260 bps) and found no significant difference in accuracy (SUP 5Khz 400 bps). In conclusion, the evaluation of ONT Q20+ nanopore sequencing chemistry demonstrated its potential as an alternative for rapid and comprehensive bacterial genome analysis in outbreak investigations. However, further research, verification studies, and optimization efforts are needed to address the observed limitations to adopt and fully realize the impact of nanopore sequencing on public health outcomes and more efficient responses to foodborne disease threats.

genomics↗

bioRxiv2021

Genomic comparison of eight closed genomes of multidrug resistant Salmonella enterica strains isolated from broiler farms and processing plants in Trinidad and Tobago

Salmonella enterica is an important foodborne pathogen worldwide. We used long and short read sequencing to close genomes of eight multidrug resistant (MDR) Salmonella enterica strains, belonging to serovars Infantis (2), Albany, Oranienburg, I 4,[5],12:i:-, Javiana, Schwarzengrund, and Kentucky from broiler chicken farms and processing plants in Trinidad and Tobago. They also belonged to seven different sequence types (STs- 32, 292, 1510, 19, 24, and 96). Seven of the strains demonstrated multi-drug resistance with the presence of at least three AMR genes. Three isolates contained the quinolone resistance gene qnrB19 in plasmids (CFSAN103840, CFSAN103854, and CFSAN103872). The extended spectrum {beta}-lactamase genes blaCTX-M-65 (CFSAN103796) and blaTEM-1 (CFSAN103852) were detected in this study. The genomes closed in this study will be useful for future source tracking and outbreak investigations in Trinidad and Tobago and worldwide.

ecology↗

bioRxiv2020

Choice of library preparation and its effects on sequence quality, genome assembly, and precise in silico prediction of virulence genes in shiga toxin producing Escherichia coli

Whole genome sequencing (WGS) provides essential public health information and is used worldwide for pathogen surveillance, epidemiology, and source tracking. The sequencing of foodborne pathogens is commonly performed with Illumina sequencing chemistry to obtain data with high accuracy. The choice of library preparation method for highly complex organisms is very critical and can affect the final data output. The majority of Illumina sequencing platforms use rapid library preparation such as Nextera XT (transposon-based technology) (Illumina San Diego, CA), but this preparation has the potential to miss randomly distributed segments of genomes that might be important for downstream analyses. The Illumina Nextera DNA Prep library preparation kit, the successor of Nextera XT, shows better overall coverage of the complete genome. This study compared the quality of sequence data generated using Nextera XT and Nextera DNA Prep kits for DNA library preparation on an Illumina MiSeq, using a set of 30 O121:H19 shiga-toxin positive Escherichia coli strains isolated from flour during a 2016 outbreak. The performance of the two kits were evaluated using several metrics including sequencing quality, assembly quality, uniformity of genome coverage, and virulence gene identification. Overall, the results showed that in all of the analysed metrics, the Nextera DNA Prep kit performed outstanding in comparison to Nextera XT. The Nextera DNA Prep kit allowed for comprehensive detection of all virulence genes, which is of extremely high importance for making an educated assessment of the virulence potential of Escherichia coli. This comprehensive side-by-side comparison will be of significance for those interested in improving their sequencing workflow for STECs and the determination of health risks using WGS data.

microbiology↗

bioRxiv2020

Haloargentinum marplatensis gen. nov., sp. nov., a novel extremely halophilic bacterium isolated from salted-ripened anchovy (Engraulis anchoita)

A facultative aerobic, Gram-negative, motile, non-endospore forming and extremely halophilic bacterium, strain 11aii{intcal}, isolated from salted-ripened anchovy, was examined using a polyphasic approach to characterize and clarify its phylogenetic and taxonomic position. Sequences of the 16S rRNA gene revealed close relationships to species of the genera Lentibacillus and Virgibacillus (94.2% similarity). The organism grew optimally in the presence of 20-35 % NaCl. The major fatty acids of strain 11aii{intcal} were C16:0 (42.1%) and anteiso-C15:0 (31.2%) and also presented iso-C16:0 (11.0%), anteiso-C17:0 (10.4%) and C18:0 (5.2%). Based on data presented here, strain 11aii{intcal} is considered to represent a novel genus and species, for which the name Haloargentinum marplatensis gen. nov. sp. nov. is proposed with the strain 11aii{intcal} as type strain.

microbiology↗

bioRxiv2020

Precision long-read metagenomics sequencing for food safety by detection and assembly of Shiga toxin-producing Escherichia coli in irrigation water

Shiga toxin-producing Escherichia coli (STEC) contamination of agricultural water might be an important factor to recent foodborne illness and outbreaks involving leafy greens. Whole genome sequencing generation of closed bacterial genomes plays an important role in source tracking. We aimed to determine the limits of detection and classification of STECs by qPCR and nanopore sequencing using enriched irrigation water artificially contaminated with E. coli O157:H7 (EDL933). We determined the limit of STEC detection by qPCR to be 30 CFU/reaction, which is equivalent to 105 CFU/ml in the enrichment. By using Oxford Nanopores EPI2ME WIMP workflow and de novo assembly with Flye followed by taxon classification with a k-mer analysis software (Kraken), E. coli O157:H7 could be detected at 103 CFU/ml (68 reads) and a complete fragmented E. coli O157:H7 metagenome-assembled genome (MAG) was obtained at 105-108 CFU/ml. Using a custom script to extract the E. coli reads, a completely closed MAG was obtained at 107-108 CFU/ml and a complete, fragmented MAG was obtained at 105-106 CFU/ml. In silico virulence detection for E. coli MAGs for 105-108 CFU/ml showed that the virulotype was indistinguishable from the spiked E. coli O157:H7 strain. We further identified the bacterial species in the un-spiked enrichment, including antimicrobial resistance genes, which could have important implications to food safety. We propose this workflow could be used for detection and complete genomic characterization of STEC from a complex microbial sample and could be applied to determine the limit of detection and assembly of other foodborne bacterial pathogens. IMPORTANCEFoodborne illness caused by Shiga toxin-producing E. coli (STEC) ranges in severity from diarrhea to hemolytic uremic syndrome and produce-related incidence is increasing. The pervasive nature of E. coli requires not only detection, but also a complete genome to determine potential pathogenicity based on stx and eae genes, serotype, and other virulence factors. We have developed a pipeline to determine the limits of nanopore sequencing for STECs in a metagenomic sample. By utilizing the current qPCR in the FDA Bacteriological Analytical Manual (BAM) Chapter 4A, we can quantify the amount of STEC in the enrichment and then sequence and classify the STEC in less than half the time as current protocols that require a single isolate. These methods have wide implications for food safety, including decreased time to STEC identification during outbreaks, characterization of the microbial community, and the potential to use these methods to determine the limits for other foodborne pathogens.

genomics↗

bioRxiv2020

Genomic analysis of the diversity, antimicrobial resistance and virulence potential of Campylobacter jejuni and Campylobacter coli strains from a private health care center in Central Chile

Campylobacter jejuni and Campylobacter coli are the leading cause of human gastroenteritis in the industrialized world and an emerging threat in developing countries. The incidence of campylobacteriosis in South America is greatly underestimated, mostly due to the lack of adequate diagnostic methods. Accordingly, there is limited genomic and epidemiological data from this region. In the present study, we performed a genome-wide analysis of the genetic diversity, virulence, and antimicrobial resistance of the largest collection of clinical C. jejuni and C. coli strains from Chile available to date (n=81), collected in 2017-2019 in Santiago, Chile. This culture collection accounts for over a third of the genome sequences available of clinical strains from South America. cgMLST analysis identified high genetic diversity as well as 13 novel STs and alleles in both C. jejuni and C. coli. Pangenome and virulome analyses showed a differential distribution of virulence factors, including both plasmid and chromosomally encoded T6SSs and T4SSs. Resistome analysis predicted widespread resistance to fluoroquinolones, but low rates of erythromycin resistance. This study provides valuable genomic and epidemiological data and highlights the need for further genomic epidemiology studies in Chile and other South American countries to better understand molecular epidemiology and antimicrobial resistance of this emerging intestinal pathogen. AUTHOR SUMMARYCampylobacter is the leading cause of bacterial gastroenteritis worldwide and an emerging and neglected pathogen in South America. In this study, we performed an in-depth analysis of the genome sequences of 69 C. jejuni and 12 C. coli clinical strains isolated from Chile, which account for over a third of the sequences from clinical strains available from South America. We identified a high genetic diversity among C. jejuni strains and the unexpected identification of clade 3 C. coli strains, which are infrequently isolated from humans in other regions of the world. Most strains harbored the virulence factors described for Campylobacter. While [~]40% of strains harbored mutation in the gyrA gene described to confer fluoroquinolone resistance, very few strains encoded the determinants linked to macrolide resistance, currently used for the treatment of campylobacteriosis. Our study contributes to our knowledge of this important foodborne pathogen providing valuable data from South America.

microbiology↗

bioRxiv2019

Nanopore sequencing for fast determination of plasmids, phages, virulence markers, and antimicrobial resistance genes in Shiga toxin-producing Escherichia coli

Whole genome sequencing can provide essential public health information. However, it is now known that widely used short-read methods have the potential to miss some randomly-distributed segments of genomes. This can prevent phages, plasmids, and virulence factors from being detected or properly identified. Here, we compared assemblies of three complete STEC O26:H11 genomes from two different sequence types (ST21 and 29), each acquired using the MiSeq-Nextera XT, MinION nanopore-based sequencing, and Pacific Biosciences (PacBio) sequencing. Each closed genome consisted of a single chromosome, approximately 5.7 Mb for CFSAN027343, 5.6 Mb for CFSAN027346, and 5.4 MB for CFSAN027350. However, short-read WGS using MiSeq-Nextera failed to identify some virulence genes in plasmids and on the chromosome, both of which were detected using the long-read platforms. Results from long-read MinION and PacBio allowed us to identify differences in plasmid content: a single 88 kb plasmid in CFSAN027343; a 157kb plasmid in CFSAN027350; and two plasmids in CFSAN027346 (one 95 Kb, one 72 Kb). These data enabled rapid characterization of the virulome, detection of antimicrobial genes, and composition/location of Stx phages. Taken together, positive correlations between the two long-read methods for determining plasmids, virulome, antimicrobial resistance genes, and phage composition support MinION sequencing as one accurate and economical option for closing STEC genomes and identifying specific virulence markers.

genomics↗

bioRxiv2018

Virulence gene profiles and phylogeny of Shiga toxin-positive Escherichia coli strains isolated from FDA regulated foods during 2010-2017

Illnesses caused by Shiga toxin-producing Escherichia coli (STECs) can be life threatening, such as hemolytic uremic syndrome (HUS). The STECs most frequently identified by USDAs Microbiological Data Program (MDP) carried toxin gene subtypes stx1a and/or stx2a. Here we describe the genome sequences of 331 STECs isolated from foods regulated by the FDA 2010-2017, determining their genomic identity, serotype, sequence type, virulence potential, and prevalence of antimicrobial resistance. Isolates were selected from the MDP archive, routine food testing by field labs (ORA), food testing by a contract company, and our laboratory (ORS). Only 276 (83%) were confirmed as STECs by in silico analysis. Foods from which STECs were recovered included cilantro (6%), spinach (25%), lettuce (11%), and flour (9%). Phylogenetic analysis using core genome MLST revealed these STEC genomes were highly variable, with some clustering associated with ST types and serotypes. We detected 95 different sequence types (ST); several ST were previously associated with HUS: ST21 and ST29 (O26:H11), ST11 (O157:H7), ST33 (O91:H14), ST17 (O103:H2), and ST16 (O111:H-). in silico virulome analyses showed ~ 51% of these strains were potentially pathogenic [besides stx gene they also carried eae (25%) or 26% subA (26%)]. Virulence gene prevalence was also determined: stx1 only (19%) -variants a and c; stx2 only (66%) - variants a, b, c, d, e, and g; and stx1/sxt2 (15%). Our data form a new WGS database that can be used to support food safety investigations and monitor the recurrence/emergence of E. coli in foods.\n\nImportanceShiga toxin-producing Escherichia coli (STECs) are associated with foodborne outbreaks worldwide; however, surveillance has not previously included genomic analyses for phylogenetics, prevalence, or potential virulence. We constructed the first genomic database of isolates from FDA-regulated foods to help monitor the emergence of new pathogenic STECs. Although only ~30 STECs were isolated per year, 50% of these carried markers associated with pathogenesis either a combination of eae plus stx, or subA plus stx. Moreover, those strains also carried virulence genes associated with severe illnesses. Here we showed that WGS enabled comparisons across isolates to establish phylogeny, help in identification of antibiotic resistance by monitoring the presence of antimicrobial resistance genes, and determined the presence of known virulence genes that have been linked with illnesses. Future food safety investigations will benefit from improved source tracking and risk assessments made possible by these analyses and new WGS database.

microbiology↗

bioRxiv2017

Parallel evolution of two clades of a major Atlantic endemic Vibrio parahaemolyticus pathogen lineage by independent acquisition of related pathogenicity islands

Shellfish-transmitted Vibrio parahaemolyticus infections have recently increased from locations with historically low disease incidence, such as the Northeast United States (US). This change coincided with a bacterial population shift towards human pathogenic variants occurring in part through the introduction of several Pacific native lineages (ST36, ST43 and ST636) to near-shore areas off the Atlantic coast of the Northeast US. Concomitantly, ST631 emerged as a major endemic pathogen. Phylogenetic trees of clinical and environmental isolates indicated that two clades diverged from a common ST631 ancestor, and in each of these clades, a human pathogenic variant evolved independently through acquisition of distinct Vibrio pathogenicity islands (VPaI). These VPaI differ from each other and bear little resemblance to hemolysin-containing VPaI from isolates of the pandemic clonal complex. Clade I ST631 isolates either harbored no hemolysins, or contained a chromosome I-inserted island we call VPaI{beta} that encodes a type three secretion system (T3SS2{beta}) typical of Trh hemolysin-producers. The more clinically prevalent and clonal ST631 clade II had an island we call VPaI{gamma} that encodes both tdh and trh and that was inserted in chromosome II. VPaI{gamma} was derived from VPaI{beta} but with some additional acquired elements in common with VPaI carried by pandemic isolates, exemplifying the mosaic nature of pathogenicity islands. Genomics comparisons and amplicon assays identified VPaI{gamma}-type islands containing tdh inserted adjacent to the ure cluster in the three introduced Pacific and most other emergent lineages. that collectively cause 67% of Northeast US infections as of 2016.\n\nIMPORTANCEThe availability of three different hemolysin genotypes in the ST631 lineage provided a unique opportunity to employ genome comparisons to further our understanding of the processes underlying pathogen evolution. The fact that two different pathogenic clades arose in parallel from the same potentially benign lineage by independent VPaI acquisition is surprising considering the historically low prevalence of community members harboring VPaI in waters along the Northeast US Coast that could serve as the source of this material. This illustrates a possible predisposition of some lineages to not only acquire foreign DNA but also to become human pathogens. Whereas the underlying cause for the expansion of V. parahaemolyticus lineages harboring VPaI{gamma} along the US Atlantic coast and spread of this element to multiple lineages that underlies disease emergence is not known, this work underscores the need to define the environment factors that favor bacteria harboring VPaI in locations of emergent disease.

evolutionary biology↗