Biology subjects

Carroll, L. M.

Publications and source records attributed to Carroll, L. M..

26 records · Page 2Linked to original sources

bioRxiv2021

Accurate de novo identification of biosynthetic gene clusters with GECCO

Biosynthetic gene clusters (BGCs) are enticing targets for (meta)genomic mining efforts, as they may encode novel, specialized metabolites with potential uses in medicine and biotechnology. Here, we describe GECCO (GEne Cluster prediction with COnditional random fields; https://gecco.embl.de), a high-precision, scalable method for identifying novel BGCs in (meta)genomic data using conditional random fields (CRFs). Based on an extensive evaluation of de novo BGC prediction, we found GECCO to be more accurate and over 3x faster than a state-of-the-art deep learning approach. When applied to over 12,000 genomes, GECCO identified nearly twice as many BGCs compared to a rule-based approach, while achieving higher accuracy than other machine learning approaches. Introspection of the GECCO CRF revealed that its predictions rely on protein domains with both known and novel associations to secondary metabolism. The method developed here represents a scalable, interpretable machine learning approach, which can identify BGCs de novo with high precision.

bioinformatics↗

bioRxiv2020

No Assembly Required: Using BTyper3 to Assess the Congruency of a Proposed Taxonomic Framework for the Bacillus cereus group with Historical Typing Methods

The Bacillus cereus group, also known as B. cereus sensu lato (s.l.), is a species complex comprising numerous closely related lineages, which vary in their ability to cause illness in humans and animals. The classification of B. cereus s.l. isolates into species-level taxonomic units is essential for facilitating communication between and among microbiologists, clinicians, public health officials, and industry professionals, but is not always straightforward. A recently proposed genomospecies-subspecies-biovar taxonomic framework aims to provide a standardized nomenclature for this species complex but relies heavily on whole-genome sequencing (WGS), a technology with limited accessibility. It thus is unclear whether popular, low-cost typing methods (e.g., single- and multi-locus sequence typing) remain congruent with the proposed taxonomy. Here, we characterize 2,231 B. cereus s.l. genomes using a combination of in silico (i) average-nucleotide identity (ANI)-based genomospecies assignment, (ii) ANI-based subspecies assignment, (iii) seven-gene multi-locus sequence typing (MLST), (iv) panC group assignment, (v) rpoB allelic typing, and (vi) virulence factor detection. We show that sequence types (STs) assigned using MLST can be used for genomospecies assignment, and we provide a comprehensive list of ST/genomospecies associations. For panC group assignment, we show that an adjusted, eight-group framework is largely congruent with the proposed eight-genomospecies taxonomy and resolves incongruencies observed in the historical seven-group framework among isolates assigned to panC Groups II, III, and VI. We additionally provide a list of loci that capture the topology of the whole-genome B. cereus s.l. phylogeny that may be used in future sequence typing efforts. For researchers with access to WGS, MLST, and/or panC data, we showcase how our recently released software, BTyper3 (https://github.com/lmc297/BTyper3), can be used to assign B. cereus s.l. isolates to taxonomic units within this proposed framework with little-to-no user intervention or domain-specific knowledge of B. cereus s.l. taxonomy. We additionally outline a novel method for assigning B. cereus s.l. genomes to pseudo-gene flow units within proposed genomospecies. The results presented here highlight the backwards-compatibility and accessibility of the proposed taxonomic framework and illustrate that WGS is not a necessity for microbiologists who want to use the proposed taxonomy effectively.

microbiology↗

bioRxiv2020

Cereulide synthetase acquisition and loss events within the evolutionary history of Group III Bacillus cereus sensu lato facilitate the transition between emetic and diarrheal foodborne pathogen

Cereulide-producing members of Bacillus cereus sensu lato (B. cereus s.l.) Group III, also known as "emetic B. cereus", possess cereulide synthetase, a plasmid-encoded, non-ribosomal peptide synthetase encoded by the ces gene cluster. Despite the documented risks that cereulide-producing strains pose to public health, the level of genomic diversity encompassed by "emetic B. cereus" has never been evaluated at a whole-genome scale. Here, we employ a phylogenomic approach to characterize Group III B. cereus s.l. genomes which possess ces (ces-positive) alongside their closely related ces-negative counterparts to (i) assess the genomic diversity encompassed by "emetic B. cereus", and (ii) identify potential ces loss and/or gain events within the evolutionary history of the high-risk and medically relevant sequence type (ST) 26 lineage often associated with emetic foodborne illness. Using all publicly available ces-positive Group III B. cereus s.l. genomes and the ces-negative genomes interspersed among them (n = 150), we show that "emetic B. cereus" is not clonal; rather, multiple lineages within Group III harbor cereulide-producing strains, all of which share a common ancestor incapable of producing cereulide (posterior probability [PP] 0.86-0.89). The ST 26 common ancestor was predicted to have emerged as ces-negative (PP 0.60-0.93) circa 1904 (95% highest posterior density [HPD] interval 1837.1-1957.8) and first acquired the ability to produce cereulide before 1931 (95% HPD 1893.2-1959.0). Three subsequent ces loss events within ST 26 were observed, including among isolates responsible for B. cereus s.l. toxicoinfection (i.e., "diarrheal" illness). Importance"B. cereus" is responsible for thousands of cases of foodborne disease each year worldwide, causing two distinct forms of illness: (i) intoxication via cereulide (i.e., "emetic" syndrome) or (ii) toxicoinfection via multiple enterotoxins (i.e., "diarrheal" syndrome). Here, we show that "emetic B. cereus" is not a clonal, homogenous unit that resulted from a single cereulide synthetase gain event followed by subsequent proliferation; rather, cereulide synthetase acquisition and loss is a dynamic, ongoing process that occurs across lineages, allowing some Group III B. cereus s.l. populations to oscillate between diarrheal and emetic foodborne pathogen over the course of their evolutionary histories. We also highlight the care that must be taken when selecting a reference genome for whole-genome sequencing-based investigation of emetic B. cereus s.l. outbreaks, as some reference genome selections can lead to a confounding loss of resolution and potentially hinder epidemiological investigations.

microbiology↗

bioRxiv2020

Comparative genomics reveals different population structures associated with host and geographic origin in antimicrobial-resistant Salmonella enterica

Genetic variation in a pathogen, including the causative agent of salmonellosis, Salmonella enterica, can occur as a result of eco-evolutionary forces triggered by dissimilarities of ecological niches. Here, we applied comparative genomics to study 90 antimicrobial resistant (AMR) S. enterica isolates from bovine and human hosts in New York state and Washington state to understand host- and geographic-associated population structure. Results revealed distinct presence/absence profiles of functional genes and pseudogenes (e.g., virulence genes) associated with bovine and human isolates. Notably, bovine isolates contained significantly more transposase genes but fewer transposase pseudogenes than human isolates, suggesting the occurrence of large-scale transposition in genomes of bovine and human isolates at different times. The high correlation between transposase genes and AMR genes, as well as plasmid replicons, highlights the potential role of horizontally transferred transposons in promoting adaptation to antibiotics. By contrast, a number of potentially geographic-associated single-nucleotide polymorphisms (SNPs), rather than geographic-associated genes, were identified. Interestingly, 38% of these SNPs were in genes annotated as cell surface protein-encoding genes, including some essential for antibiotic resistance and host colonization. Overall, different evolutionary forces and limited recent inter-population transmission appear to shape AMR S. enterica population structure in different hosts and geographic origins. Originality/Significance StatementSalmonella enterica, which is the causative agent of salmonellosis, poses a growing public health concern due to the emergence and spread of antimicrobial resistant (AMR) strains. The mechanisms underlying the population structure associated with different hosts and geographic origins of AMR S. enterica are underexplored due to limited genome-wide studies assessing the impact of ecological niches on genetic variations. By employing comparative genomics, our study provided insights into the genomic profiles of AMR S. enterica associated with two distinct hosts and two distant geographic locations, improving the mechanistic understanding of how bacterial population structure is shaped by different ecological niches. Our findings have broad implications for elucidating the impact of ecological and evolutionary forces on the adaptation, antimicrobial resistance, and pathogenicity of bacteria. Also, specific genetic markers we identified may help predict host or geographic origin of AMR Salmonella isolates, which could benefit the source tracking (e.g., host and geographic origins) of human disease cases and contamination events caused by AMR S. enterica.

microbiology↗

bioRxiv2019

Proposal of a taxonomic nomenclature for the Bacillus cereus group which reconciles genomic definitions of bacterial species with clinical and industrial phenotypes

The Bacillus cereus group comprises numerous closely related species, including bioterrorism agent B. anthracis, foodborne pathogen B. cereus, and biopescticide B. thuringiensis. Differentiating organisms capable of causing illness or death from those used in industry is essential for risk assessment and outbreak preparedness. However, current species definitions facilitate species-phenotype incongruencies, particularly when horizontally acquired genes are responsible for a phenotype. Using all publicly available B. cereus group genomes (n = 2,231), we show that current genomospecies definitions lead to overlapping species clusters, and that an average nucleotide identity (ANI) genomospecies threshold of {approx}92.5 reflects a natural gap in core genome similarity. We propose a taxonomy for the B. cereus group which accounts for (i) genomospecies using separable species clusters formed at a threshold of {approx}92.5 ANI, and (ii) phenotypes relevant to public health and industry. We anticipate that the proposed nomenclature will remain interpretable to clinicians, without sacrificing genomic species definitions, which can in turn aid in pathogen surveillance, early detection of emerging, high-risk genotypes, and outbreak preparedness. Furthermore, the nomenclatural framework outlined here serves as a model for genomics-based bacterial taxonomy which moves beyond arbitrarily set genomospecies thresholds, while maintaining congruence with phenotypes and historically important species names.

microbiology↗

bioRxiv2019

Identification of novel mobilized colistin resistance gene mcr-9 in a multidrug-resistant, colistin-susceptible Salmonella enterica serotype Typhimurium isolate using a combination of high-throughput, in silico screening and functional analysis

Mobilized colistin resistance (mcr) genes are plasmid-borne genes that confer resistance to colistin, an antibiotic used to treat severe bacterial infections. To date, eight known mcr homologues have been described (mcr-1 to -8). Here, we describe mcr-9, a novel mcr homologue, detected in a Salmonella enterica serotype Typhimurium (S. Typhimurium) genome using an in silico approach, followed by experimental functional analysis. The amino acid sequence of mcr-9, detected in a multidrug resistant (MDR) S. Typhimurium strain isolated from a human patient in Washington State in 2010, most closely resembled mcr-3, aligning with 64.5% amino acid identity and 99.5% coverage using translated nucleotide blast. The S. Typhimurium strain was tested for phenotypic resistance to colistin and was found to be sensitive at the 2 mg/L European Committee on Antimicrobial Susceptibility Testing breakpoint under the tested conditions. To determine whether it was capable of conferring resistance to colistin when expressed in a heterologous host, mcr-9 was cloned in colistin-susceptible Escherichia coli NEB5 under an IPTG-induced promoter. Expression of mcr-9 conferred resistance to colistin in E. coli NEB5 at 1, 2, and 2.5 mg/L colistin, albeit at a lower level when compared to mcr-3. Pairwise comparisons of the predicted protein structures associated with all nine mcr homologues (Mcr-1 to -9) revealed that Mcr-9, Mcr-3, and Mcr-7 share a high degree of similarity at the structural level. The results of our approach indicate that mcr-9 is capable of conferring phenotypic resistance to colistin in Enterobacteriaceae and should be immediately considered when monitoring plasmid-mediated colistin resistance. Importance: Colistin is a last-resort antibiotic that is used to treat severe infections caused by MDR and extensively drug resistant (XDR) bacteria. The World Health Organization (WHO) has designated colistin as a Highest Priority Critically Important Antimicrobial for human medicine (WHO, Critically Important Antimicrobials for Human Medicine, 5th Revision, 2017), as it is often one of the only therapies available for treating serious bacterial infections in critically ill patients. Plasmid-borne mcr genes that confer resistance to colistin pose a threat to public health at an international scale, as they can be transmitted via horizontal gene transfer and have the potential to spread globally. Therefore, the establishment of a complete reference of mcr genes that can be used to screen for plasmid-mediated colistin resistance is essential for developing effective control strategies.

microbiology↗

bioRxiv2019

Bacillus clarus sp. nov. is a new Bacillus cereus group species isolated from soil

Bacillus cereus group or B. cereus sensu lato (s.l.), is comprised of Gram-positive spore-forming, rod-like bacteria that are widespread in natural environments. Although the species in this group are known to be highly related in terms of phenotypic characteristics, they display different levels of pathogenicity. Biochemical assays are therefore considered to be insufficient for accurate taxonomic classification of B. cereus group species. To facilitate accurate taxonomic classification and associated prediction of pathogenic potential, we have conducted comparative genomic analyses of publicly available genome assemblies of B. cereus group isolates. Through that, we found that an isolate previously known as B. mycoides ATCC 21929 was sufficiently distant from valid and effective type strains to be considered a putative new species. We have conducted biochemical and bioinformatic characterization of strain ATCC 21929 that had been isolated from soil in Papua New Guinea. Strain ATCC 21929 most closely resembles B. paramycoides NH24A2T, producing ANIb and DDH values of 86.70% and 34.1%, respectively. Phenotypically, isolate ATCC 21929 does not possess cytochrome c oxidase activity, and is able to grow at a range of temperatures 15{degrees}C - 43{degrees}C and at a range of pH 6 - 9. With regards to fatty acid composition, this isolate has iso-C17:0 in highest abundance. We propose the strain ATCC 21929T (=PS00077AT = PS00077BT = PSU-0922T = BHPT) as a new species named Bacillus clarus sp. nov. to facilitate accurate taxonomic classification of B. cereus group isolates.

microbiology↗

bioRxiv2018

Characterization of emetic and diarrheal Bacillus cereus strains from a 2016 foodborne outbreak using whole-genome sequencing: addressing the microbiological, epidemiological, and bioinformatic challenges

The Bacillus cereus group comprises multiple species capable of causing emetic or diarrheal foodborne illness. Despite being responsible for tens of thousands of illnesses each year in the U.S. alone, whole-genome sequencing (WGS) has not been routinely employed to characterize B. cereus group isolates from foodborne outbreaks. Here, we describe the first WGS-based characterization of isolates linked to an outbreak caused by members of the B. cereus group. In conjunction with a 2016 outbreak traced to a supplier of refried beans served by a fast food restaurant chain in upstate New York, a total of 33 B. cereus group strains were obtained from human cases (n =7) and food samples (n = 26). Emetic (n = 30) and diarrheal (n = 3) isolates were most closely related to B. paranthracis (clade III) and B. cereus sensu stricto (clade IV), respectively. WGS indicated that the 30 emetic isolates (24 and 6 from food and humans, respectively) were closely-related and formed a well-supported clade relative to publicly-available emetic clade III genomes with an identical sequence type (ST 26). When compared to publicly-available emetic clade III ST 26 B. cereus group genomes, the 30 emetic clade III isolates from this outbreak differed from each other by a mean of 8.3 to 11.9 core single nucleotide polymorphisms (SNPs), while differing from publicly-available genomes by a mean of 301.7 to 528.0 core SNPs, depending on the SNP calling methodology used. Using a WST-1 cell proliferation assay, the strains isolated from this outbreak had only mild detrimental effects on HeLa cell metabolic activity compared to reference diarrheal strain B. cereus ATCC 14579. Based on both WGS and epidemiological data, we hypothesize that the outbreak was a single source outbreak caused by emetic clade III B. cereus belonging to the B. paranthracis species. In addition to showcasing how WGS can be used to characterize B. cereus group strains linked to a foodborne outbreak, we also discuss potential microbiological and epidemiological challenges presented by B. cereus group outbreaks, and we offer recommendations for analyzing WGS data from the isolates associated with them.

microbiology↗