Biology subjects

Kovac, J.

Publications and source records attributed to Kovac, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Environmental Microbial Community Signatures Associated with Listeria spp. Detection in German Meat Processing Facilities

Listeria monocytogenes can persist in niches of meat processing environments despite routine cleaning and disinfection. Its persistence may depend not only on stress tolerance but also on interactions with resident microbial communities, which may promote or inhibit survival. However, these ecological relationships remain poorly understood. We combined 16S rRNA V3/V4 amplicon sequencing, culture-based detection, and multilocus sequence typing (MLST) to characterize microbial communities in six German meat processing facilities over one year. We examined associations among community structure, sampling sites (drains and food-contact surfaces), and the occurrence of Listeria spp., including L. monocytogenes. Microbial communities were dominated by core genera typical of food-processing environments, particularly Pseudomonas spp. and Acinetobacter spp., but differed significantly among facilities (PERMANOVA, p = 0.001; pairwise R{superscript 2} = 0.023-0.079), indicating facility-specific communities. Culture-based analyses detected Listeria spp. in 51 of 370 environmental samples (13.8%), mainly from drains (44/51, 86.3%). L. monocytogenes was detected in five of six facilities, with 19 of 21 isolates originating from drains (90.5%). MLST of 74 typeable L. monocytogenes isolates revealed high diversity, comprising 21 sequence types across 15 clonal complexes, with lineage II predominating (86.5%). Overall microbial community composition was significantly associated with Listeria spp. and L. monocytogenes presence (PERMANOVA, p = 0.001; R{superscript 2} = 0.0137 and 0.0083). In drains, ASVs assigned to Acinetobacter, Rhizorhapis, and Vagococcus species showed positive associations with Listeria spp.-positive samples. Together these findings suggest that drains are key ecological niches for Listeria spp. and that associated taxa may indicate drain communities linked to Listeria spp. recovery. IMPORTANCEListeria monocytogenes is a major foodborne pathogen that can persist in meat processing environments despite routine cleaning and disinfection. Resident microbial communities may influence its survival, but longitudinal studies linking those communities with culture-based Listeria spp. detection remain limited. Here, we characterized microbial communities in six German meat processing facilities over 1 year using 16S rRNA gene amplicon sequencing and culture-based Listeria spp. detection, and MLST of L. monocytogenes isolates. We identified facility-specific microbial communities, identified floor drains as key niches for Listeria spp., and observed repeated recovery of different L. monocytogenes sequence types across facilities. In drains, ASVs assigned to the genera Acinetobacter, Rhizorhapis, and Vagococcus species were positively associated with culture-positive samples, identifying candidate taxa that may reflect microbial conditions associated with Listeria spp. recovery. These findings highlight the importance of considering not only whether Listeria spp. are detected, but also the resident microbial communities that may support their fitness.

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

Soilless system design impacts the diversity and composition of microbiota

Controlled environment agriculture (CEA), including soilless farming systems, is rapidly expanding as a strategy to improve food security and resource efficiency. However, limited information is available on how different soilless farming system designs influence microbial populations relevant to plant health and food safety. This study investigated the effects of soilless growing systems and growing season on aerobic plate counts (APC) and bacterial community composition in nutrient solution and on bok choy (Brassica rapa subsp. chinensis) leaves. Five soilless systems, deep water culture (DWC), Kratky (KR), nutrient film technique (NFT), ebb and flow (EF), and drip irrigation (DI), were evaluated across fall and spring growing seasons. Soilless system type significantly influenced APC in nutrient solution, with the DI system consistently exhibiting the highest counts across both seasons. Increased nutrient solution pH was negatively associated with APC, whereas temperature did not significantly affect bacterial concentrations. In contrast, APC on bok choy leaves were not significantly influenced by system type, season, pH, or temperature. Bacterial community composition in nutrient solution was strongly shaped by season, soilless system type, sampling day, and temperature, as determined by 16S rRNA V4 amplicon sequencing. Microbial diversity varied primarily by system type, with limited influence of pH or temperature. Core microbiota analysis identified a small subset of taxa that persisted across systems and seasons, with Acidovorax detected in all samples. We found that soilless system design and seasonal conditions strongly influence microbial load and community structure in nutrient solution, providing a foundation for developing system-specific microbial management strategies. ImportanceUnderstanding factors that shape microbial community composition in soilless farming systems is critical for optimizing plant health, system productivity, and food safety. Microbial communities influence nutrient cycling, biofilm formation, and pathogen survival, all of which affect the ecological stability and performance of these systems. By identifying how system design, seasonal variation, and environmental conditions influence shifts in microbial populations, targeted strategies can be developed to promote beneficial microorganisms and mitigate risks associated with pathogens. This knowledge contributes to advancing safe and sustainable soilless farming practices that can meet the growing demand for fresh produce grown in controlled environments.

microbiology↗

bioRxiv2025

Single nucleotide polymorphisms associated with cytotoxicity of Bacillus cereus group strains in Caco-2 cells

Bacillus cereus sensu lato (s.l.) encompasses strains with diverse impacts, ranging from foodborne illness and anthrax to beneficial applications in agriculture and industry. While the risk of anthrax and emetic intoxication can be reliably predicted by the presence of specific virulence genes, predicting diarrheal foodborne illness risk based solely on enterotoxin gene presence has proven unreliable. In this study, we evaluated cytotoxicity against Caco-2 human gut cells using a diverse collection of B. cereus s.l. isolates representing all eight panC phylogenetic groups and conducted genomic analyses to identify predictive markers of cytotoxicity. Isolates from panC groups I, IV, and V exhibited significantly higher cytotoxicity compared to other groups, although individual isolates from other panC groups have also been linked to illness. Logistic and random forest regression models revealed that while the presence of enterotoxin genes was a sensitive indicator of cytotoxicity, it lacked specificity. Logistic regression analysis identified 21 nonsynonymous single nucleotide polymorphisms (SNPs) within enterotoxin (Nhe and Hbl) gene sequences that were more effective predictors of cytotoxicity, providing higher specificity with comparable sensitivity. These SNPs achieved accuracy and precision values exceeding 0.7. Random forest models highlighted the importance of panC group, enterotoxin gene SNPs, and the presence of the full hbl operon as key predictors of cytotoxicity. The strong sensitivity, specificity, and biological relevance of these SNPs position them as promising markers for improving strain-based risk assessment of B. cereus s.l. IMPORTANCEEnterotoxin genes have been associated with B. cereus sensu lato (s.l.) diarrheal foodborne illness; however, their mere presence in a genome is an unreliable predictor of an isolates cytotoxicity towards human gut epithelial cells. To improve food safety risk assessment, more specific markers of cytotoxicity are required. In this study, we identified nonsynonymous SNPs within the coding sequences of the enterotoxins Nhe and Hbl. These SNPs offer potential targets for rapid molecular tests to identify B. cereus s.l. isolates with an elevated food safety risk due to their capacity to inflict cytotoxic damage on human gut epithelial cells. Implementation of such markers upon validation could improve consumer safety while reducing food waste.

microbiology↗

bioRxiv2024

Ecology of prophage-like elements in Bacillus subtilis at global and local geographical scale

Prophages account for a substantial part of most bacterial genomes, but the impacts on hosts remain poorly understood. Here, we combined computational and laboratory experiments to explore the abundance, distribution, and activity of prophage elements in Bacillus subtilis. NCBI database genome sequences and isolates from 1 cm3 riverbank soil samples were analyzed to provide insights at global and local geographical scales, respectively. Most prophages in wild B. subtilis isolates were related to mobile genetic elements previously identified in laboratory strains. Some large groups of prophages were closely related to completely uncharacterized yet functional Bacillus phages, or completely unknown. As certain prophage groups were unique to local isolates, we explored factors influencing prophages within a single genome. Phylogenetic relatedness was a slightly better predictor of host prophage repertoire than geographical origin. We show that cryptic phages can play a major role in acquisition and/or maintenance of other prophage elements both via strong antagonism or by co-dependence. Laboratory experiments showed that most predicted prophages may be cryptic, since they failed to induce under DNA-damaging stress conditions. Interestingly, the magnitude of stress responses remained proportional to the total number of prophage elements predicted, suggesting their importance in host physiology. This study highlights the diversity, integration patterns, and co-occurrence of prophages in B. subtilis and their potential impact on host evolution and physiology. Understanding these dynamics provides insight into bacterial genome evolution and prophage-host interactions, laying the groundwork for future experimental studies on the roles of phages in the ecology and evolution of this bacterial species.

microbiology↗

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

kmerDB: A Database Encompassing the Set of Genomic and Proteomic Sequence Information for Each Species

The rapid decline in sequencing cost has enabled the generation of reference genomes and proteomes for a growing number of organisms. However, at the present time, there is no established repository that provides information about organism-specific genomic and proteomic sequences of certain lengths, also known as kmers, that are either present or absent in each genome or proteome. In this article, we present kmerDB, a database accessible through an interactive web interface that provides kmer based information from genomic and proteomic sequences in a systematic way. kmerDB currently contains 202,340,859,107 base pairs and 19,304,903,356 amino acids, spanning 45,785 and 22,386 reference genomes and proteomes, respectively, as well as 14,658,776 and 149,264,442 genomic and proteomic species-specific sequences, termed quasi-primes. Additionally, we provide access to 5,186,757 nucleic and 214,904,089 peptide sequences that are absent from every genome and proteome, termed primes. kmerDB features a user-friendly interface offering various search options and filters for easy parsing and searching. The service is available at: www.kmerdb.com.

genomics↗

bioRxiv2022

Context matters: environmental microbiota of ice cream processing facilities affects the inhibitory performance of two lactic acid bacteria against Listeria monocytogenes

Pathogenic L. monocytogenes may inhabit dairy processing environments, increasing the risk for cross-contamination of foods. Using biocontrol microorganisms that inhibit or outcompete L. monocytogenes to complement sanitation of dairy processing facilities may enhance the control of L. monocytogenes. However, it remains unknown whether the resident microbiota of dairy processing facilities affects the antilisterial activity of biocontrol strains. Here, two lactic acid bacteria (LAB) strains (Enterococcus PS01155 and PS01156) were tested for their biocontrol potential in the context of microbiomes collected from three ice cream processing facilities (A, B, and C). Antilisterial ability was assessed by co-culturing LABs with 8-L. monocytogenes strains in the presence of microbiota for 3 days at 15{degrees}C, followed by quantification of the most probable number of attached L. monocytogenes. L. monocytogenes concentration increased by 0.38{+/-}0.77 log10 MPN/sample in treatments containing microbiota from facility A, while it decreased by 0.99{+/-}1.13 and 2.54{+/-}0.84 log10 MPN/sample in treatments with microbiota from facilities B and C, respectively. The attachment of LAB to an abiotic surface was assessed by co-culturing LABs in with the microbiomes at 15{degrees}C for 3 days, followed by characterization of attached microbiota composition using amplicon sequencing. All samples containing microbiomes from facilities A and B had high relative abundance of Pseudomonas, while samples with facility C microbiome had high relative abundance of Enterococcus. Overall, we show that microbiota composition of ice cream processing facilities affected the antilisterial ability of LABs. IMPORTANCEAntilisterial lactic acid bacteria strains had been proposed as biological pathogen control agents for application in food processing environments. However, the effect of resident food processing environment microbiota on the performance on antilisterial lactic acid bacteria strains is poorly understood. Our study shows that the composition of the microbiota collected from ice cream processing facilities environmental surfaces can affect the attachment and inhibitory effect of lactic acid bacteria strains against L. monocytogenes. Further studies are therefore needed to evaluate whether individual microbial taxa affect antilisterial properties of lactic acid bacteria strains and to characterize the underlying mechanisms.

microbiology↗

bioRxiv2022

Conditional forest models built using metagenomic data could accurately predict Salmonella contamination in Northeastern streams

The use of water contaminated with Salmonella for produce production contributes to foodborne disease burden. To reduce human health risks, there is a need for novel, targeted approaches for assessing the pathogen status of agricultural water. We investigated the utility of water microbiome data for predicting Salmonella contamination of streams used to source water for produce production. Grab samples were collected from 60 New York streams in 2018 and tested for Salmonella. Separately, DNA was extracted from the samples and used for Illumina shotgun metagenomic sequencing. Reads were trimmed and used to assign taxonomy with Kraken2. Conditional forest (CF), regularized random forest (RRF), and support vector machine (SVM) models were implemented to predict Salmonella contamination. Model performance was determined using 10-fold cross-validation repeated 10 times to quantify area under the curve (AUC) and Kappa score. Taxa identified as the most informative for accurately predicting Salmonella contamination based on conditional variable importance were compared to taxa identified by ALDEx2 as being differentially abundant between Salmonella-positive and - negative samples. CF models outperformed the other two algorithms based on AUC (0.82 - CF, 0.76 - RRF, 0.67 - SVM) and Kappa score (0.41- CF, 0.38 - RRF, 0.19 - SVM). CF and differential abundance tests both identified Aeromonas (VI = 0.32) and Tabrizicola (VI = 0.12) as the two most informative taxa for predicting Salmonella contamination. The taxa identified in this study warrant further investigation as indicators of Salmonella contamination in Northeastern freshwater streams. IMPORTANCEUnderstanding the associations between surface water microbiome composition and the presence of foodborne pathogens, such as Salmonella, can facilitate the identification of novel indicators of Salmonella contamination. This study assessed the utility of microbiome data and three machine learning algorithms for predicting Salmonella contamination of Northeastern streams. The research reported here both expanded the knowledge on the microbiome composition of surface waters and identified putative novel indicators (i.e., Aeromonas and Tabrizicola) for Salmonella in Northeastern streams. These putative indicators warrant further research to assess whether they are consistent indicators of Salmonella for regions, waterways, and years not represented in the dataset used in this study.

microbiology↗

bioRxiv2022

Short term tomato consumption alters the pig gut microbiome towards a more favorable profile

Diets rich in fruits and vegetables have been shown to exert positive effects on the gut microbiome. However, little is known about the specific effect of individual fruits or vegetables on gut microbe profiles. This study aims to elucidate the effects of tomato consumption on the gut microbiome, as tomatoes account for 22% of vegetable consumption in Western diets, and their consumption has been associated with positive health outcomes. Using piglets as a physiologically relevant model of human metabolism, 20 animals were assigned either to a control or tomato powder supplemented diet (both macronutrient matched and isocaloric) for 14 days. The microbiome was sampled rectally at three time points: day 0 (baseline), day 7 (midpoint), and at day 14 (end of study). DNA was sequenced using shotgun metagenomics, and reads were annotated using MG-RAST. There were no differences in body weight or feed intake between our two treatment groups. There was a microbial shift which included a higher ratio of Bacteroidota to Bacillota (formerly known as Bacteroidetes and Firmicutes, respectively) and higher alpha-diversity in tomato-fed animals, indicating a shift to a more desirable phenotype. Analyses at both the phyla and genera levels showed global microbiome profile changes (PERMANOVA P [≤] 0.05) over time, but not with tomato consumption. These data suggest that short-term tomato consumption can beneficially influence the gut microbial profile, warranting further investigation in humans. IMPORTANCEThe composition of the microorganisms in the gut is a contributor to overall health, prompting the development of strategies to alter the microbiome composition. Studies have investigated the role of the diet on the microbiome, as it is a major modifiable risk factor contributing to health; however, little is known about the causal effects of consumption of specific foods on the gut microbiota. A more complete understanding of how individual foods impact the microbiome will enable more evidence-based dietary recommendations for long-term health. Tomatoes are of interest as the most consumed non-starchy vegetable and a common source of nutrients and phytochemicals across the world. This study aimed to elucidate the effect of short-term tomato consumption on the microbiome, using piglets as a physiologically relevant model to humans. We found that tomato consumption can positively affect the gut microbial profile, which warrants further investigation in humans.

microbiology↗

bioRxiv2021

Comparative analysis of Bacillus cereus group isolates' resistance using disk diffusion and broth microdilution and the correlation between resistance phenotypes and genotypes

A collection of 85 Bacillus cereus group isolates were screened for phenotypic resistance to nine antibiotics using disk diffusion and broth microdilution. The broth microdilution antimicrobial results were interpreted using the CLSI M45 breakpoints for Bacillus spp. Due to the lack of Bacillus spp. disk diffusion breakpoints, the results obtained with the disk diffusion assay were interpreted using the CLSI M100 breakpoints for Staphylococcus spp. We identified significant (p < 0.05) discrepancies in resistance interpretation between the two methods for ampicillin, gentamicin, rifampicin, tetracycline, and trimethoprim/sulfamethoxazole. Antimicrobial resistance genes were detected using unassembled and assembled whole-genome sequences with Ariba and Abricate, respectively, to assess the sensitivity and specificity for predicting phenotypic resistance based on the presence of antimicrobial resistance genes. We found antimicrobial resistance gene presence to be a poor indicator for phenotypic resistance, calling for further investigation of mechanisms underlying antimicrobial resistance in the B. cereus group. Genes with poor sensitivity and/or specificity, as determined based on broth microdilution results included rph (rifampicin, 0%, 95%), mph genes (erythromycin, 0%, 96%), and all van genes (vancomycin, 100%, 35%). However, Bc (ampicillin, 64%, 100%) and tet genes (tetracycline, 67%, 100%) were highly specific, albeit moderately sensitive indicators of phenotypic resistance based on broth microdilution results. Only beta-lactam resistance genes (Bc, BcII, and blaTEM) were highly sensitive (94%) and specific (100%) markers of resistance to ceftriaxone based on the disk diffusion results, providing further evidence of these beta-lactams role in nonsusceptibility of Bacillus cereus group isolates to ceftriaxone. IMPORTANCEBacillus cereus group includes human pathogens that can cause severe infections requiring antibiotic treatment. Screening of environmental and food isolates for antimicrobial resistance can provide insight into what antibiotics may be more effective therapeutic options based on the lower prevalence of resistance. Currently, interpretation of antimicrobial susceptibility testing results using the disk diffusion method is complicated by the fact that there are no standard disk diffusion resistance breakpoints defined for Bacillus spp. Hence, the breakpoints for Staphylococcus are often used in research studies. By comparing the results of disk diffusion interpreted using the Staphylococcus spp. breakpoints against broth microdilution interpreted using Bacillus spp. breakpoints, this study demonstrated that disk diffusion results interpretation with Staphylococcus spp. breakpoints are inconsistent. This study also provides new insight into the poor associations between antimicrobial resistance genotypes and phenotypes for the B. cereus group.

microbiology↗

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

Dynamic Laser Speckle Imaging meets Machine Learning to enable Rapid Antibacterial Susceptibility Testing (DyRAST)

Rapid antibacterial susceptibility testing (RAST) methods which measure change of a bacterial phenotype in response to a given treatment are of significant importance in healthcare, as they can assist care-givers in timely administration of the right treatment. Various RAST techniques have been reported for tracking bacterial phenotypes, such as size, shape, motion, and metabolic activity. However, they still require bulky and expensive instruments (which hinders their application in resource-limited environments) and/or utilize labeling reagents (which can interfere with antibiotics and add to cost). Furthermore, the existing ultra-rapid methods do not address possible adaptation of gradual adaptation of bacteria to antibiotics, which can lead to false interpretation of resistance when using ultra-rapid methods. In this work, we present a RAST approach leveraging machine learning analysis of time-resolved dynamic laser speckle imaging (DLSI) results to accurately predict the minimum inhibitory concentration (MIC) of a model strain of Escherichia coli in 60 minutes, compared to 6 hours using the currently FDA-approved phenotype-based RAST technique. To demonstrate the DLSI performance, we studied the effect of a {beta}-lactam ampicillin and an aminoglycoside gentamicin on Escherichia coli strain K-12. DLSI captures change of bacterial motion/division in response to treatment. The machine learning algorithm was trained and validated using the overnight results of gold standard, broth microdilution method. Empowered by machine learning, DyRAST can predict MIC with high accuracy comparable to gold standard methods through a voting strategy.

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

Intra-species DNA exchange: Bacillus subtilis prefers sex with less related strains

B. subtilis is a surface-dwelling bacterium that is competent for natural transformation. Genetically distinct B. subtilis swarms form a boundary upon encounter, mediated by a fast-evolving molecular kin discrimination (KD) system. We here demonstrate that KD-mediated swarm antagonisms in B. subtilis promote transformation-mediated horizontal gene transfer between strains of low relatedness. Gene transfer is largely uni-directional and the result of the induction of competence of the recipient strain by the donor strain is linked to a sigW-mediated stress response. More closely related strains, which in theory would experience more efficient recombination due to increased sequence homology, did not upregulate transformation upon encounter, demonstrating that social interactions can override mechanistic barriers to horizontal gene transfer. KD-mediated competence in response to the encounter of distinct neighbouring strains could maximize the probability of efficient incorporation of novel alleles and genes that have proved to function in a genomically and ecologically similar context.

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.

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