Biology subjects

Carroll, L. M.

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

At least 19 recordsLinked to original sources

bioRxiv2026

Hiding in plain sight: rare Bacillus cereus group species are present among unassembled metagenomic samples in public databases

Abstract The Bacillus cereus group is a complex of closely related species, which vary in terms of their geographic spread, industrial utility, and pathogenic potential. In addition to encompassing well-known species represented by thousands of genomes (e.g., bioterrorism agent B. anthracis, foodborne pathogen B. cereus, biopesticide B. thuringiensis), the B. cereus group houses "rare" species discovered/first sequenced over a decade ago. Until recently, four rare species (B. manliponensis, B. bingmayongensis, B. gaemokensis, "B. clarus") were each represented by a single unique genome; as such, virtually nothing is known about these species beyond their type strains. We hypothesized that additional representatives of rare B. cereus group species may already be present in public databases as yet-unassembled metagenomes. To this end, we used species-specific markers to identify 7 metagenomic samples in NCBI's Sequence Read Archive, which harbored B. manliponensis, B. bingmayongensis, or B. gaemokensis. Metagenomic assembly and binning yielded an isolate-quality B. manliponensis metagenome-assembled genome (MAG) from avocado orchard soil in Israel, plus a partial B. bingmayongensis MAG from commercial spinach spiked with Shiga toxin-producing Escherichia coli. For rare species represented by >1 unique genome, we compared our novel MAGs to all available genomes and identified strain- and lineage-specific genomic elements (e.g., sphingomyelinase C and hemolysin BL homologs, variably present in B. manliponensis and B. bingmayongensis, respectively). Altogether, our study indicates that some rare B. cereus group species may be more common and geographically widespread than previously thought, and highlights the utility of culture-independent methods for recovering rare/under-isolated taxa. Impact Statement Individual B. cereus group strains vary in terms of their pathogenic potential and industrial utility. While some strains cause illness in humans and/or animals (e.g., anthrax, emetic intoxication, diarrheal toxicoinfection, non-gastrointestinal illnesses), other strains have important biotechnological applications (e.g., biocontrol, plant growth promotion, production of antimicrobials and other natural products). Because many virulence- and biotechnological-related phenotypes are strain-dependent (rather than species-dependent), phenotypes associated with an individual strain (e.g., a species type strain) cannot be extrapolated to an entire B. cereus group species. Within this context, rare B. cereus group species represent a major gap in our understanding of B. cereus group evolution, ecology, and pathogenicity, as until 2021, each rare species was represented solely by its type strain genome. Here, we aggregate all publicly available genomes/MAGs of rare B. cereus group species and supplement them with their first novel representatives since 2024. Our efforts bring the total number of unique B. manliponensis and B. bingmayongensis genomes/MAGs to >2, allowing us to conduct the first-ever phylogeographic survey and strain-level assessment of functional potential for each species. Overall, our study represents a critical first step toward understanding rare B. cereus group species beyond their type strains.

microbiology↗

bioRxiv2026

The insecticidal potential of Bacillus cereus group strains from insect-dense regions of the United Kingdom

Entomopathogenic Bacillus cereus group members (e.g. B. thuringiensis) are arguably the most important source of biopesticides globally and are widely used for industrial-scale crop protection. However, the application of biopesticidal B. cereus group strains is currently threatened by increasing resistance among insects, necessitating the development of novel insecticides. Here, we used whole-genome sequencing to characterize 13 novel B. cereus group strains from sites with high-density insect populations, including six isolates from an insect cadaver. Overall, diverse B. cereus group members were observed, encompassing three panC groups (II, IV and VI) and seven sequence types. Five strains possessed insecticidal Cry toxin-encoding genes with predicted target ranges that included insect species present at the respective sampling locations. Moreover, the genomes were computationally mined for biosynthetic gene clusters (BGCs; clusters of genes governing secondary metabolite synthesis). In total, 274 BGCs were observed, of which the majority (170 BGCs; 62.04%) did not cluster with known BGCs. Taken together, our study indicates that sampling B. cereus group bacteria from insect-rich ecological niches may yield Cry toxins against the self-same insects, as well as novel and unexplored BGCs, suggesting that targeted sampling efforts could generate compound leads for biopesticide development.

microbiology↗

bioRxiv2026

Antimicrobial Resistance Profiling and Phenotypic Characterization of Archived Clinical Bacillus paranthracis Strains

Bacillus paranthracis was formally defined as a species in 2017, after decades of carrying the name "emetic B. cereus" based on cereulide production and clustering within B. cereus sensu lato phylogenetic group III. Commonly associated with foodborne intoxication, reports rarely link B. paranthracis to non-foodborne clinical illness. As such, the new taxonomy and close resemblance of the name to the biothreat pathogen Bacillus anthracis cause confusion in diagnostic and public health settings. To address this issue, B. paranthracis clinical strains (n=20) from the CDC collection were tested with microbiological methods used for identification of B. anthracis and antimicrobial susceptibility testing. Some B. paranthracis phenotypes were similar to B. anthracis, however others were inconsistent across strains. Like B. anthracis: 3 strains tested capsule positive, 5 were non-hemolytic on blood agar, and 9 non-motile. All B. paranthracis strains were resistant to gamma phage lysis, which differentiated them from B. anthracis. Treatment regimens for B. paranthracis infections are not well established, as antimicrobial therapy is not indicated for emetic intoxication caused by B. paranthracis. Notably, six B. paranthracis strains had elevated minimal inhibitory concentrations to anthrax-recommended antibiotics: one for ciprofloxacin, three for doxycycline and tetracycline, and two for clindamycin. Rapid MinION sequencing was assessed for antimicrobial resistance detection prediction but had limited value when using PiMA v.1. These microbiological observations and susceptibility profiles of B. paranthracis expand our understanding of this pathogen, strengthening our ability to differentiate this bacterium from B. anthracis to improve diagnosis and patient outcomes. IMPORTANCEThis study describes in vitro characterization of 20 archived clinical strains of B. paranthracis, an opportunistic pathogen identified more frequently in recent reports. Our findings highlight phenotypic differences and similarities between B. paranthracis and B. anthracis using standard microbiological methods and drug susceptibility profiling. We also assess a rapid B. anthracis specific MinION long read genome sequencing workflow with B. paranthracis. This report highlights the overlapping morphological features shared by B. paranthracis and B. anthracis to improve future laboratory diagnosis and strengthen anthrax preparedness. This article will effectively reach an audience of public health professionals and microbiologists strengthening anthrax preparedness.

microbiology↗

bioRxiv2026

NheABC is a pH-dependent cytotoxin that contributes to the virulence of Bacillus cereus

Bacillus cereus is a Gram-positive bacterium widely distributed in food, soil, and plants. It is a spore-forming, facultative anaerobic bacterium known as a food-borne opportunistic human pathogen responsible for causing gastrointestinal and non-gastrointestinal infections, including wound-associated infections. In the present study, we report that among single-component, bi-component, and tripartite alpha pore-forming toxin (-PFT) producing bacteria, the tripartite NheABC toxin produced by B. cereus induced the maximum cell death in infected epithelial cells. Similar to its effects in 2D monolayers, NheABC exhibited potent cell toxicity in 3D spheroids and intestinal organoids, targeting their cell membrane and mitochondria. Moreover, using erythrocytes as a model system, we found that the cytolytic activity of NheABC is pH-dependent, and was markedly reduced at acidic pH (5.5). The pH-dependent biological activity of NheABC was further confirmed by a liposome leakage assay. Importantly, NheABC enhanced the colonization of B. cereus in a non-gastrointestinal murine wound infection model. Overall, our study highlights the role of pH in regulating NheABC-mediated cytotoxicity in mammalian cells, which may lead to the development of novel therapeutic strategies for managing B. cereus gastrointestinal and non-gastrointestinal infections.

microbiology↗

bioRxiv2025

Large-scale insights into the biosynthetic potential of the Bacillus cereus group

Bacterial secondary metabolites (SMs) are a critical source of natural product-derived drugs. However, SM discovery efforts have focused overwhelmingly on Actinomycetes, potentially overlooking other key producers. Here, we explore the biosynthetic potential of the Bacillus cereus group, an underexplored complex of SM producers. Using a combined rule- and machine learning-based approach, we mine an unprecedented number of B. cereus group genomes (n = 9,744) for SM-producing biosynthetic gene clusters (BGCs; n = 200,196). Notably, 158,678 B. cereus group BGCs (78.2%) did not cluster with previously described BGCs, suggesting new chemical scaffolds to be explored. B. pseudomycoides was particularly prolific in terms of its SM production potential (30.8 BGC families/genome, Kruskal-Wallis p < 0.0001), and we identify a previously uncharacterized, B. pseudomycoides-unique peptide. Overall, our study represents the largest survey of B. cereus group biosynthetic potential to date and posits the complex as an under-queried SM resource.

microbiology↗

bioRxiv2025

Scalable and interpretable secretion system annotation with Sismis

Secretion systems play critical roles in bacterial growth, survival, and pathogenesis. Genes encoding secretion system components often co-occur together as gene clusters in bacterial (meta)genomes. However, existing tools for secretion system annotation are unable to utilize genomic context to make predictions and are unable to detect secretion systems of novel architecture. Here, we present Sismis (secretion system discovery tool; https://github.com/lmc297/Sismis), a scalable, interpretable, machine learning-based tool, which detects and classifies single-locus secretion systems in bacterial (meta)genomes with high accuracy (test set area-under-the-curve [AUC] values of 0.71 and 0.92 for precision-recall [PR] and receiver operating characteristic [ROC] curves, respectively). When applied to {approx}700k prokaryotic (meta)genomes, Sismis identifies 747, 439 total secretion systems comprising 15, 612 major secretion system families, >80% of which contain no previously known/annotated secretion systems. To facilitate further exploration of these data, we present the Sismis Atlas (https://sismis.microbe.dev/), an interactive secretion system database, which we use to identify a largely uncharacterized cluster of secretion systems with tight adherence (Tad) pili-like characteristics. Altogether, Sismis and its companion atlas enable accurate and interpretable secretion system annotation, exploration, and discovery at an unprecedented scale. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/675188v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@89dec2org.highwire.dtl.DTLVardef@17fa652org.highwire.dtl.DTLVardef@18064b9org.highwire.dtl.DTLVardef@54deaa_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Scalable single-cell metagenomic analysis with Bascet and Zorn

Single-cell metagenomic sequencing (scMetaG) can provide maximum-resolution insights into complex microbial communities. However, existing bioinformatic tools are not equipped to handle the massive amounts of data generated by novel high-throughput scMetaG methods. Here, we present a bioinformatic toolkit for complete, end-to-end scMetaG analysis: (i) Bascet, a command-line suite designed to scale to massive scMetaG datasets ([≥]1 million cells); (ii) Zorn, an R package/workflow manager that enables reproducible scMetaG data analysis, exploration, and visualization (http://zorn.henlab.org/). Enabled by recent advances in droplet microfluidics, we use Bascet and Zorn to develop and optimize a high-throughput scMetaG method on a ten-species mock community. To showcase their utility on a real-world sample, we use Bascet and Zorn to characterize a human saliva sample, generating single-amplified genomes (SAGs) from >10k prokaryotic cells. Overall, Bascet and Zorn enable reproducible scMetaG analysis, allowing users to query microbiomes at unprecedented resolution and scale.

microbiology↗

bioRxiv2025

Fast, flexible gene cluster family delineation with IGUA

Prokaryotic genomes harbor a variety of functional elements encoded as contiguous multi-gene clusters, with biosynthetic gene clusters (BGCs, genetic determinants of secondary metabolite biosynthesis) serving as a notable example. In a typical workflow, BGCs are clustered into Gene Cluster Families (GCFs), units that group BGCs encoding similar biosynthetic pathways together. However, existing methods cannot readily scale to massive datasets and cannot be used for GCF delineation tasks beyond BGC clustering. Here, we present IGUA (Iterative Gene clUster Analysis; https://github.com/zellerlab/IGUA), a scalable, flexible GCF delineation method for genomic segments with multi-gene architectures. On a BGC clustering task, IGUA is [≥]10x faster than the state-of-the-art (BiG-SCAPE/BiG-SLiCE), without sacrificing accuracy. To highlight its scalability, we use IGUA to cluster >2.8 million BGCs from {approx}1 million prokaryotic genomes in <18 hours (n = 2,829,071 BGCs to 56,960 GCFs). To showcase its utility beyond BGC clustering, we use IGUA to cluster (i) secretion systems and (ii) prophages into GCFs (n = 10,576 and 356,776 gene clusters to 2,744 and 213,699 GCFs, respectively). Overall, IGUA represents a versatile GCF delineation tool with unmatched computational efficiency and flexibility, enabling (meta)genomic mining applications at unprecedented scales. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/654203v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1f27216org.highwire.dtl.DTLVardef@203ccborg.highwire.dtl.DTLVardef@7766c8org.highwire.dtl.DTLVardef@fcd4b5_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2025

PyOrthoANI, PyFastANI, and Pyskani: a suite of Python libraries for computation of average nucleotide identity

SummaryThe average nucleotide identity (ANI) metric has become the gold standard for prokaryotic species delineation in the genomics era. The most popular ANI algorithms are available as command-line tools and/or web applications, making it inconvenient or impossible to incorporate them into bioinformatic workflows, which utilize the popular Python programming language. Here, we present PyOrthoANI, PyFastANI, and Pyskani, Python libraries for three popular ANI computation methods. ANI values produced by PyOrthoANI, PyFastANI, and Pyskani are virtually identical to those produced by OrthoANI, FastANI, and skani, respectively. All three libraries integrate seamlessly with BioPython, making it easy and convenient to use, compare, and benchmark popular ANI algorithms within Python-based workflows. Availability and ImplementationSource code is open-source and available via GitHub (PyOrthoANI, https://github.com/althonos/orthoani; PyFastANI, https://github.com/althonos/pyfastani; Pyskani, https://github.com/althonos/pyskani). Supplementary InformationSupplementary data are available on bioRxiv.

bioinformatics↗

bioRxiv2024

Exposure assessment suggests some cytotoxic Bacillus cereus group genotypes can grow over 3 logs in HTST milk throughout the shelf life at temperature abuse conditions

Cytotoxic Bacillus cereus group strains are common causes of foodborne illness including diarrhea. However, our ability to assess food safety risks associated with the exposure to cytotoxic B. cereus group strains via contaminated food is limited due to the lack of predictive tools. In this study, we experimentally quantified the growth of 17 cytotoxic B. cereus group strains, representing six phylogenetic groups, in skim milk broth and used the growth data to develop an exposure assessment model. While none of the tested strains showed detectable growth in HTST milk at 4 or 6{degrees}C, 15 of the 17 strains showed growth at 10{degrees}C, 1 of the 17 strains showed growth at 8{degrees}C, and all strains grew at [≥]14{degrees}C. Growth data for 16 strains allowed us to generate linear secondary growth models, which were then used to develop the exposure assessment model. We simulated a five-stage supply chain with up to 35 consumer storage days, as that was the timing when distinguishable variations in percent milk containers over 105 CFU/m with different B. cereus genotypes were observed. When the initial contamination level of the HTST milk is set at an average of 100 CFU/mL, the model predicts that, on consumer home storage day 21 and 35, 2.81{+/-}0.66 and 4.13{+/-}2.53 % (mean {+/-} standard deviation) of the milk containers would exceed B. cereus group concentrations of 105 CFU/mL; these data represent the average across all strains. Sensitivity analysis showed that variation in the input parameter Q0, the initial physiological state of cells, has the largest effect on models prediction for 1 of 4 group II isolates, 1 of 6 group IV isolates and both group V isolates, suggesting the need to better characterize the growth parameters of these isolates. What-if scenario analysis showed that increased mean and variability in storage temperature at the consumers home both have substantial influence on final predicted B. cereus group concentration in milk containers. This model introduces an initial tool designed to facilitate risk-based food safety decision making for products that are contaminated with low B. cereus group levels.

microbiology↗

bioRxiv2024

Comparison of the performance of multiple whole-genome sequence-based tools for the identification of Bacillus cereus sensu stricto biovar Thuringiensis

The Bacillus cereus sensu stricto (s.s.) species comprises strains of biovar Thuringiensis (Bt) known for their bioinsecticidal activity, as well as strains with foodborne pathogenic potential. Bt strains are identified (i) based on the production of insecticidal crystal proteins also known as Bt toxins or (ii) based on the presence of cry, cyt, and vip genes, which encode Bt toxins. Multiple bioinformatics tools have been developed for the detection of crystal protein-encoding genes based on whole-genome sequencing (WGS) data. However, the performance of these tools is yet to be evaluated using phenotypic data. Thus, the goal of this study was to assess the performance of four bioinformatics tools for the detection of crystal protein-encoding genes. The accuracy of sequence-based identification of Bt was determined in reference to phenotypic microscope-based screening for production of crystal proteins. A total of 58 diverse B. cereus s.l. strains isolated from clinical, food, environmental, and commercial biopesticide products were underwent WGS. Isolates were examined for crystal protein production using phase contrast microscopy. Crystal protein-encoding genes were detected using BtToxin_Digger, BTyper3, IDOPS, and Cry_processor. Out of 58 isolates, the phenotypic production of crystal proteins was confirmed for 18 isolates. Specificity and sensitivity of Bt identification based on sequences were 0.85 and 0.94 for BtToxin_Digger, 0.97 and 0.89 for BTyper3, 0.95 and 0.94 for IDOPS, and 0.88 and 1.00 for Cry_processor, respectively. Cry_processor predicted crystal protein production with highest specificity, and BtToxin_Digger and IDOPS predicted crystal protein production with the highest sensitivity. Three out of four tested bioinformatic tools performed well overall, with IDOPS achieving both high sensitivity and specificity (>0.90). IMPORTANCEBacillus cereus s.s. biovar Thuringiensis (Bt) is used as an organic biopesticide. It is differentiated from the foodborne pathogen Bacillus cereus s.s. by the production of insecticidal crystal proteins. Thus, reliable genomic identification of biovar Thuringiensis is necessary to ensure food safety and facilitate risk assessment. This study assessed the accuracy of WGS-based identification of Bt compared to phenotypic microscopy-based screening for crystal protein production. Multiple bioinformatics tools were compared to assess their performance in predicting crystal protein production. Among them, IDOPS performed best overall at WGS- based Bt identification.

bioinformatics↗

bioRxiv2023

A community-curated, global atlas of Bacillus cereus sensu lato genomes for epidemiological surveillance

The ability to cause foodborne illness, anthrax, and other infections has been attributed to numerous lineages within Bacillus cereus sensu lato (s.l.). However, existing pathogen surveillance databases facilitate dangerous pathogen misidentifications when applied to B. cereus s.l., potentially hindering outbreak or bioterrorism attack response efforts. To address this, we developed BTyperDB (www.btyper.app), an atlas of B. cereus s.l. genomes with standardized, community-curated metadata. BTyperDB aggregates all publicly available B. cereus s.l. genomes (including >2,600 previously unassembled genomes) with novel genomes donated by laboratories around the world, nearly doubling the number of publicly available B. cereus s.l. genomes. To showcase its utility for pathogen surveillance, we use BTyperDB to identify emerging anthrax toxin- and capsule-harboring lineages. Overall, our study provides insight into the epidemiology of an under-studied group of emerging pathogens and highlights the benefits of inclusive, community-driven metadata FAIRification efforts.

microbiology↗

bioRxiv2023

Genus-Wide Genomic Characterization of Macrococcus: Insights into Evolution, Population Structure, and Functional Potential

Macrococcus species have been isolated from a range of mammals and mammal-derived food products. While they are largely considered to be animal commensals, Macrococcus spp. can be opportunistic pathogens in both veterinary and human clinical settings. This study aimed to provide insight into the evolution, population structure, and functional potential of the Macrococcus genus, with an emphasis on antimicrobial resistance (AMR) and virulence potential. All high-quality, publicly available Macrococcus genomes (n = 104, accessed 27 August 2022), plus six South African genomes sequenced here (two strains from bovine clinical mastitis cases and four strains from beef products), underwent taxonomic assignment (using four different approaches), AMR determinant detection (via AMRFinderPlus), and virulence factor detection (using DIAMOND and the core Virulence Factor Database). Overall, the 110 Macrococcus genomes were of animal commensal, veterinary clinical, food-associated (including food spoilage), and environmental origins; five genomes (4.5%) originated from human clinical cases. Notably, none of the taxonomic assignment methods produced identical results, highlighting the potential for Macrococcus species misidentifications. The most common predicted antimicrobial classes associated with AMR determinants identified across Macrococcus included macrolides, beta-lactams, and aminoglycosides (n = 81, 61, and 44 of 110 genomes; 73.6, 55.5, and 40.0%, respectively). Genes showing homology to Staphylococcus aureus exoenzyme aureolysin were detected across multiple species (using 90% coverage, n = 40 and 77 genomes harboring aureolysin-like genes at 60% and 40% amino acid [AA] identity, respectively). Staphylococcus aureus Panton-Valentine leucocidin toxin-associated lukF-PV and lukS-PV homologs were identified in eight M. canis genomes ([≥]40% AA identity, >85% coverage). Using a method that delineates populations using recent gene flow (PopCOGenT), two species (M. caseolyticus and M. armenti) were composed of multiple within-species populations. Notably, M. armenti was partitioned into two populations, which differed in functional potential (e.g., one harbored beta-lactamase family, type II toxin-antitoxin system, and stress response proteins, while the other possessed a Type VII secretion system; PopCOGenT P < 0.05). Overall, this study leverages all publicly available Macrococcus genomes in addition to newly sequenced genomes from South Africa to identify genomic elements associated with AMR or virulence potential, which can be queried in future experiments.

microbiology↗

bioRxiv2022

More than mcr: Canonical Plasmid- and Transposon-Encoded Mobilized Colistin Resistance (mcr) Genes Represent a Subset of Phosphoethanolamine Transferases

Mobilized colistin resistance genes (mcr) may confer resistance to colistin, a last-resort, critically important antimicrobial for human health. mcr can often be transmitted horizontally (e.g., via mobile genetic elements); however, mcr encode phosphoethanolamine transferases (PET) closely related to chromosomally encoded, intrinsic lipid modification enzymes (e.g., EptA, EptB, CptA). To explore the genetic diversity of mcr within the context of intrinsic lipid modification PET, we identified 9,836 non-redundant protein accession numbers associated with mcr-like genes, representing a total of 69,814 mcr-like genes present across 256 bacterial genera. We subsequently identified 125 unique, putative novel mcr-like genes encoded on the same contig as a plasmid replicon and other antimicrobial resistance genes. Sequence similarity and a maximum likelihood phylogeny of mcr, putative novel mcr-like genes, and intrinsic lipid modification PET-encoding genes indicated that sequence similarity is insufficient to discriminate between genes involved in colistin resistance and genes encoding intrinsic lipid modification PET. A mixed-effect model of evolution (MEME) indicated that site- and branch-specific diversifying positive selection might have played a role in the evolution of subvariants within the mcr-2 and mcr-9 families. MEME suggested that positive selection played a role in the diversification of several residues in structurally important regions, including (i) a bridging region that connects the membrane-bound and catalytic periplasmic domains, and (ii) a periplasmic loop juxtaposing the substrate entry tunnel. These residues were found to be differentially conserved in different mcr families and thus may play a role in mcr subvariant phenotypic diversity. Moreover, we found that eptA and mcr are localized within different genomic contexts. Canonical eptA are typically chromosomally encoded in an operon with a two-component regulatory system or adjacent to a TetR-type regulator. In contrast, mcr are encoded as single-gene operons or adjacent to pap2 and dgkA, which encode a PAP2 family lipid A phosphatase and diacylglycerol kinase, respectively. Our data suggest that eptA can give rise to "colistin resistance genes" through various mechanisms, including selection and diversification of the genomic context, regulatory pathways, and mobilization. These mechanisms likely altered gene expression levels and enzyme activity, allowing bona fide eptA to evolve to function in colistin resistance.

microbiology↗

bioRxiv2022

A multidrug-resistant Salmonella enterica serotype Typhimurium DT104 lineage circulating among humans and cattle in the United States lost the ability to produce pertussis-like toxin ArtAB in close temporal proximity to the global DT104 epidemic

Salmonella enterica subspecies enterica serotype Typhimurium definitive type 104 (DT104) can infect both humans and animals and is often multidrug-resistant (MDR). Previous studies have indicated that, unlike most S. Typhimurium, the overwhelming majority of DT104 strains produce pertussis-like toxin ArtAB via prophage-encoded genes artAB. However, DT104 that lack artAB have been described on occasion. Here, we identify a MDR DT104 complex lineage circulating among humans and cattle in the United States, which lacks artAB (i.e., the "U.S. artAB-negative major clade"; n = 42 genomes). Unlike most other bovine- and human-associated DT104 complex strains from the U.S. (n = 230 total genomes), which harbor artAB on prophage Gifsy-1 (n = 177), members of the U.S. artAB-negative major clade lack Gifsy-1, as well as anti-inflammatory effector gogB. The U.S. artAB-negative major clade encompasses human- and cattle-associated strains isolated from [≥]11 U.S. states over a twenty-year period. The clade was predicted to have lost artAB, Gifsy-1, and gogB circa 1985-1987 (95% highest posterior density interval 1979.0-1992.1). When compared to DT104 genomes from other world regions (n = 752 total genomes), several additional, sporadic artAB, Gifsy-1, and/or gogB loss events among clades encompassing [≤]5 genomes were observed. Using phenotypic assays that simulate conditions encountered during human and/or bovine digestion, members of the U.S. artAB-negative major clade did not differ from closely related Gifsy-1/artAB/gogB-harboring U.S. DT104 complex strains (ANOVA raw P-value > 0.05); thus, future research is needed to elucidate the roles that artAB, gogB, and Gifsy-1 play in DT104 virulence in humans and animals. Impact StatementMulti-drug resistant (MDR) Salmonella enterica serotype Typhimurium definitive type 104 (DT104) was responsible for a global epidemic among humans and animals throughout the 1990s and continues to circulate worldwide. Previous studies have indicated that the vast majority of DT104 produce pertussis-like toxin ArtAB via prophage-encoded artAB. Here, we identify a DT104 complex lineage that has been circulating among cattle and humans across [≥]11 U.S. states for over twenty years, which lacks the ability to produce ArtAB (i.e., the "U.S. artAB-negative major clade"). The common ancestor of all U.S. artAB-negative major clade members lost the ability to produce ArtAB in the 1980s; however, the reason for this loss-of-function event within this well-established pathogen remains unclear. The role that ArtAB plays in DT104 virulence remains elusive, and phenotypic assays conducted here indicate that members of the U.S. artAB-negative major clade do not have a significant advantage or disadvantage relative to closely related, Gifsy-1/artAB/gogB-harboring U.S. DT104 complex strains when exposed to stressors encountered during human and/or bovine digestion in vitro. However, ArtAB heterogeneity within the DT104 complex suggests clade-specific selection for or against maintenance of ArtAB. Thus, future studies querying the virulence characteristics of the U.S. artAB-negative major clade are needed. Data SummarySupplementary Data is available under DOI 10.5281/zenodo.7688792, with URL https://doi.org/10.5281/zenodo.7688792.

microbiology↗

bioRxiv2022

Genomic surveillance of Bacillus cereus sensu lato strains isolated from meat and poultry products in South Africa enables inter- and intra-national surveillance and source tracking

Members of the Bacillus cereus sensu lato (s.l.) species complex, also known as the B. cereus group, vary in their ability to cause illness, but are frequently isolated from foods, including meat products; however, food safety surveillance efforts that employ whole-genome sequencing (WGS) often neglect these potential pathogens. Here, WGS was used to characterize B. cereus s.l. strains (n = 25) isolated during surveillance of meat products in South Africa. Strains were collected from beef, poultry, and mixed meat products obtained from (i) retail outlets, processing plants, and butcheries across six South African provinces (n = 15, 7, and 1, respectively), and (ii) imports in cold stores (n = 2). Strains were assigned to panC Groups IV, III, II, and V (n = 18, 5, 1, and 1, respectively) and spanned multiple genomospecies, regardless of the taxonomy used. All strains possessed diarrheal toxin-encoding genes, while one sequence type 26 (ST26) strain possessed cereulide (emetic toxin) synthetase-encoding genes. No strains harbored anthrax toxin- or capsule-encoding genes. The 25 strains were partitioned into 15 lineages via in silico seven-gene multi-locus sequence typing (MLST), six of which contained multiple strains sequenced in this study, which were identical or nearly identical at the whole-genome scale. Five MLST lineages contained (nearly) identical genomes collected from two or three South African provinces; one MLST lineage contained nearly identical genomes from two countries (South Africa and the Netherlands), indicating that B. cereus s.l. can spread intra- and inter-nationally via foodstuffs. ImportanceNation-wide foodborne pathogen surveillance programs that employ high-resolution genomic methods have been shown to provide vast public health and economic benefits. However, B. cereus s.l. are often overlooked during large-scale, routine WGS efforts. Thus, to our knowledge, no studies to date have evaluated the potential utility of WGS for B. cereus s.l. surveillance and source tracking in foodstuffs. In this proof-of-concept study, we applied WGS to B. cereus s.l. strains collected via South Africas national surveillance program of domestic and imported meat products, and we provide strong evidence that B. cereus s.l. can be disseminated intra- and inter-nationally via the agro-food supply chain. Our results showcase that WGS can be used for source tracking of B. cereus s.l. in foods, although future WGS and isolate metadata collection efforts are needed to ensure that B. cereus s.l. surveillance initiatives are on par with those of other foodborne pathogens.

microbiology↗

bioRxiv2021

Monitoring the Antimicrobial Resistance Dynamics of Salmonella enterica in Healthy Dairy Cattle Populations at the Individual Farm Level Using Whole-Genome Sequencing

Livestock represent a possible reservoir for facilitating the transmission of the zoonotic foodborne pathogen Salmonella enterica to humans; there is also concern that strains can acquire resistance to antimicrobials in the farm environment. Here, we use whole-genome sequencing (WGS) to characterize Salmonella strains (n = 128) isolated from healthy dairy cattle and their associated environments on 13 New York State farms to assess the diversity and microevolution of this important pathogen at the level of the individual herd. Additionally, the accuracy and concordance of multiple in silico tools are assessed, including: (i) two in silico serotyping tools, (ii) combinations of five antimicrobial resistance (AMR) determinant detection tools and one to five AMR determinant databases, and (iii) one antimicrobial minimum inhibitory concentration (MIC) prediction tool. For the isolates sequenced here, in silico serotyping methods outperformed traditional serotyping and resolved all un-typable and/or ambiguous serotype assignments. Serotypes assigned in silico showed greater congruency with the Salmonella whole-genome phylogeny than traditional serotype assignments, and in silico methods showed high concordance (99% agreement). In silico AMR determinant detection methods additionally showed a high degree of concordance, regardless of the pipeline or database used ([≥]98% agreement between susceptible/resistant assignments for all pipeline/database combinations). For AMR detection methods that relied exclusively on nucleotide BLAST, accuracy could be maximized by using a range of minimum nucleotide identity and coverage thresholds, with thresholds of 75% nucleotide identity and 50-60% coverage adequate for most pipeline/database combinations. In silico characterization of the microevolution and AMR dynamics of each of six serotype groups (S. Anatum, Cerro, Kentucky, Meleagridis, Newport, Typhimurium/Typhimurium variant Copenhagen) revealed that some lineages were strongly associated with individual farms, while others were distributed across multiple farms. Numerous AMR determinant acquisition and loss events were identified, including the recent acquisition of cephalosporin resistance-conferring blaCMY- and blaCTX-M-type beta-lactamases. The results presented here provide high-resolution insight into the temporal dynamics of AMR Salmonella at the scale of the individual farm and highlight both the strengths and limitations of WGS in tracking zoonotic pathogens and their associated AMR determinants at the livestock-human interface.

microbiology↗

bioRxiv2021

Genomic characterization of endemic and ecdemic non-typhoidal Salmonella enterica lineages circulating among animals and animal products in South Africa

Non-typhoidal Salmonella enterica imposes a significant burden on human and animal health in South Africa. However, very little is known about lineages circulating among animals and animal products in the country on a genomic scale. Here, we used whole-genome sequencing (WGS) to characterize 63 Salmonella enterica strains (n = 18, 8, 13, and 24 strains assigned to serotypes Dublin, Hadar, Enteritidis, and Typhimurium, respectively) isolated from livestock, companion animals, wildlife, and animal products in South Africa over a 60-year period. Within-serotype phylogenies were constructed using genomes sequenced in this study, as well as publicly available genomes representative of each respective serotypes (i) global (n = 2,802 and 1,569 S. Dublin and Hadar genomes, respectively) and (ii) African (n = 716 and 343 S. Enteritidis and Typhimurium genomes, respectively) population. For S. Dublin, the approaches used here identified a largely antimicrobial-susceptible, endemic lineage circulating among humans, animals, and food in South Africa, as well as a lineage that was likely recently introduced from the United States. For S. Hadar, multiple South African lineages harboring streptomycin and tetracycline resistance-conferring genes were identified. African S. Enteritidis could be primarily partitioned into one largely antimicrobial-susceptible and one largely multidrug-resistant (MDR) clade, with South African isolates confined to the largely antimicrobial-susceptible clade. S. Typhimurium strains sequenced here were distributed across the African S. Typhimurium phylogeny, representing a diverse range of lineages, including numerous MDR lineages. Overall, this study provides insight into the evolution, population structure, and antimicrobial resistome composition of Salmonella enterica in Africa. IMPORTANCEGlobally, Salmonella enterica is estimated to be responsible for more than 93 million illnesses and 150,000 deaths annually. In Africa, the burden of salmonellosis is disproportionally high; however, WGS efforts are overwhelmingly concentrated in world regions with lower salmonellosis burdens. While WGS is being increasingly employed in South Africa to characterize Salmonella enterica, the bulk of these efforts have centered on characterizing human clinical strains. WGS data derived from non-typhoidal Salmonella enterica serotypes isolated from non-human sources in South Africa is extremely limited. To our knowledge, the genomes sequenced here represent the largest collection of non-typhoidal Salmonella enterica isolate genomes from non-human sources in South Africa to date. Furthermore, this study provides critical insights into endemic and ecdemic non-typhoidal Salmonella enterica lineages circulating among animals, foods, and humans in South Africa and showcases the utility of WGS in characterizing animal-associated strains from a world region with a high salmonellosis burden.

microbiology↗