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 the production of crystal proteins. A total of 58 diverse B. cereus sensu lato strains isolated from clinical, food, environmental, and commercial biopesticide products underwent WGS. Isolates were examined for crystal protein production using phase contrast microscopy. Crystal protein-encoding genes were detected using BtToxin_Digger, BTyper3, IDOPS (identification of pesticidal sequences), 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 the highest specificity, and BtToxin_Digger and IDOPS predicted crystal protein production with the highest sensitivity. Three out of four tested bioinformatics tools performed well overall, with IDOPS achieving high sensitivity and specificity (>0.90).IMPORTANCEStrains of Bacillus cereus sensu stricto (s.s.) biovar Thuringiensis (Bt) are used as organic biopesticides. Bt 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 whole-genome sequencing (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, identification of pesticidal sequences performed best overall at WGS-based Bt identification.

Recent paradigm shifts in the perception of the role of Bacillus thuringiensis in foodborne disease.

Plant protection products based on Bacillus thuringiensis have been used to fight agricultural pests for decades and are the world's most frequently applied biopesticide. However, there is growing concern that B. thuringiensis residues in food may occasionally cause diarrheal illness in humans. This has recently sparked a plethora of research activities and vivid discussions across the scientific community, competent authorities, and the public. To support this discussion, we provide a structured overview of the current knowledge on the role of B. thuringiensis as a causative agent of foodborne infections in humans and pinpoint research gaps that need to be addressed for improved risk assessment. We review (i) recent taxonomic changes in the B. cereus group; (ii) the role of B. thuringiensis in transforming agrosystems; and (iii) key considerations for assessing the hazard potential of B. thuringiensis strains detected in foods. We conclude that (i) the taxonomy of the B. cereus group is collapsing, (ii) B. thuringiensis based biopesticides play a key role in realizing the UN's sustainable development goals, and (iii) risk assessment needs to move from taxonomy-driven considerations to strain-specific identification of virulence and pathogenicity traits We also provide an overview of relevant risk-related data for commonly used biopesticide strains.

Population structure and virulence gene profiles of Streptococcus agalactiae collected from different hosts worldwide.

Streptococcus agalactiae is a leading cause of morbidity and mortality among neonates and causes severe infections in pregnant women and nonpregnant predisposed adults, in addition to various animal species worldwide. Still, information on the population structure of S. agalactiae and the geographical distribution of different clones is limited. Further data are urgently needed to identify particularly successful clones and obtain insights into possible routes of transmission within one host species and across species borders. We aimed to determine the population structure and virulence gene profiles of S. agalactiae strains from a diverse set of sources and geographical origins. To this end, 373 S. agalactiae isolates obtained from humans and animals from five different continents were typed by DNA microarray profiling. A total of 242 different S. agalactiae strains were identified and further analyzed. Particularly successful clonal lineages, hybridization patterns, and strains were identified that were spread across different continents and/or were present in more than one host species. In particular, several strains were detected in both humans and cattle, and several canine strains were also detected in samples from human, bovine, and porcine hosts. The findings of our study suggest that although S. agalactiae is well adapted to various hosts including humans, cattle, dogs, rodents, and fish, interspecies transmission is possible and occurs between humans and cows, dogs, and rabbits. The virulence and resistance gene profiles presented enable new insights into interspecies transmission and make a crucial contribution to the identification of suitable targets for therapeutic agents and vaccines.

Effect of food-related stress conditions and loss of agr and sigB on seb promoter activity in S. aureus.

Staphylococcal enterotoxin B (SEB) causes staphylococcal food poisoning and is produced in up to ten times higher quantities than other major enterotoxins. While Staphylococcus aureus growth is often repressed by competing flora, the organism exhibits a decisive growth advantage under some stress conditions. So far, data on the influence of food-related stressors and regulatory mutations on seb expression is limited and largely based on laboratory strains, which were later reported to harbor mutations. Therefore, the aim of this study was to investigate the influence of stress and regulatory mutations on seb promoter activity. To this end, transcriptional fusions were created in two strains, USA300 and HG003, carrying different seb upstream sequences fused to a blaZ reporter. NaCl, nitrite, and glucose stress led to significantly decreased seb promoter activity, while lactic acid stress resulted in significantly increased seb promoter activity. Loss of agr decreased seb promoter activity and loss of sigB increased promoter activity, with the magnitude of change depending on the strain. These results demonstrate that mild stress conditions encountered during food production and preservation can induce significant changes in seb promoter activity.

East and West African milk products are reservoirs for human and livestock-associated Staphylococcus aureus.

Staphylococcus aureus frequently isolated from milk products in sub-Saharan Africa (SSA) is a major pathogen responsible for food intoxication, human and animal diseases. SSA hospital-derived strains are well studied but data on the population structure of foodborne S. aureus required to identify possible staphylococcal food poisoning sources is lacking. Therefore, the aim was to assess the population genetic structure, virulence and antibiotic resistance genes associated with milk-derived S. aureus isolates from Côte d'Ivoire, Kenya and Somalia through spa-typing, MLST, and DNA microarray analysis. Seventy milk S. aureus isolates from the three countries were assigned to 27 spa (7 new) and 23 (12 new) MLST sequence types. Milk-associated S. aureus of the three countries is genetically diverse comprising human and livestock-associated clonal complexes (CCs) predominated by the CC5 (n = 10) and CC30 (n = 9) isolates. Panton-Valentine leukocidin, toxic shock syndrome toxin and enterotoxin encoding genes were predominantly observed among human-associated CCs. Penicillin, fosfomycin and tetracycline, but not methicillin resistance genes were frequently detected. Our findings indicate that milk-associated S. aureus in SSA originates from human and animal sources alike highlighting the need for an overarching One Health approach to reduce S. aureus disease burdens through improving production processes, animal care and hygienic measures.

Genetic and Phenotypic Characteristics of Staphylococcus aureus Isolates from Cystic Fibrosis Patients in Austria.

BACKGROUND: Cystic fibrosis (CF) is the most common life-limiting inherited disease in Caucasian populations. While pathological changes can be seen in various organs, morbidity and mortality are mainly related to the respiratory tract, with patients suffering from chronic bronchopulmonary infections with characteristic pathogens including Staphylococcus aureus. OBJECTIVES: To date, there is only very limited data on the genetic and phenotypic characteristics of S. aureus in CF patients. Therefore, in our study, we characterized 58 S. aureus isolates collected from CF patients in Austria by spa typing, DNA microarray profiling, as well as antimicrobial susceptibility testing in order to determine common genomic and antimicrobial resistance features. The tested strain collection exhibited high genomic diversity. RESULTS: The 58 isolates were assigned to 16 clonal complexes and 48 spa types and differed greatly regarding their virulence and resistance gene profiles. The predominant clonal complexes were MLST CC30 (22%), CC15 (16%), CC45 (14%), and CC5 (12%), complexes that are highly prevalent worldwide among S. aureus strains isolated from humans colonized or infected with S. aureus. DNA microarray profiles showed a wide variety of genes encoding antimicrobial resistance and virulence factors such as various leukocidins, haemolysins, enterotoxins, exfoliative toxins, toxic shock syndrome toxin, as well as genes involved in adhesion and immune evasion. CONCLUSIONS: While a large number of strains exhibited resistance to one or several antimicrobial agents, methicillin-resistant S. aureus was found at a low prevalence of 3% (n = 2) only. The two methicillin-resistant S. aureus isolates were assigned to CC152/t355 (SCCmecV) and CC5/t001 (SCCmecI). This is the first study to genetically characterize S. aureus isolates in CF patients in Austria.

Outbreak of staphylococcal food poisoning among children and staff at a Swiss boarding school due to soft cheese made from raw milk.

On October 1, 2014, children and staff members at a Swiss boarding school consumed Tomme, a soft cheese produced from raw cow milk. Within the following 7h, all 14 persons who ingested the cheese fell ill, including 10 children and 4 staff members. Symptoms included abdominal pain and violent vomiting, followed by severe diarrhea and fever. We aim to present this food poisoning outbreak and characterize the causative agent. The duration of the incubation period was dependent of the age of the patient: 2.5h in children under 10 yr of age, 3.5h in older children and teenagers, and 7h in adults. The soft cheese exhibited low levels of staphylococcal enterotoxin (SE) A (>6ng of SEA/g of cheese) and high levels of staphylococcal enterotoxin D (>200ng of SED/g of cheese). Counts of 10(7) cfu of coagulase-positive staphylococci per gram of cheese were detected, with 3 different Staphylococcus aureus strains being present at levels >10(6) cfu/g. The 3 strains were characterized using spa typing and a DNA microarray. An enterotoxin-producing strain exhibiting sea and sed was identified as the source of the outbreak. The strain was assigned to spa type tbl 3555 and clonal complex 8, and it exhibited genetic criteria consistent with the characteristics of a genotype B strain. This genotype comprises bovine Staph. aureus strains exclusively associated with very high within-herd prevalence of mastitis and has been described as a major contaminant in Swiss raw milk cheese. It is therefore highly likely that the raw milk used for Tomme production was heavily contaminated with Staph. aureus and that levels further increased due to growth of the organism and physical concentration effects during the cheese-making process. Only a few staphylococcal food poisoning outbreaks involving raw milk products have been described. Still, in view of this outbreak and the possible occurrence of other foodborne pathogens in bovine milk, consumption of raw milk and soft cheese produced from raw milk constitutes a health risk, particularly when young children or other members of sensitive populations are involved.

Investigation of a staphylococcal food poisoning outbreak combining case-control, traditional typing and whole genome sequencing methods, Luxembourg, June 2014.

In June 2014, a staphylococcal food poisoning outbreak occurred at an international equine sports event in Luxembourg requiring the hospitalisation of 31 persons. We conducted a microbiological investigation of patients and buffet items, a case-control study and a carriage study of catering staff. Isolates of Staphylococcus aureus from patients, food and catering staff were characterised and compared using traditional typing methods and whole genome sequencing. Genotypically identical strains (sequence type ST8, spa-type t024, MLVA-type 4698, enterotoxin A FRI100) were isolated in 10 patients, shiitake mushrooms, cured ham, and in three members of staff. The case-control study strongly suggested pasta salad with pesto as the vehicle of infection (p<0.001), but this food item could not be tested, because there were no leftovers. Additional enterotoxigenic strains genetically unrelated to the outbreak strain were found in four members of staff. Non-enterotoxigenic strains with livestock-associated sequence type ST398 were isolated from three food items and two members of staff. The main cause of the outbreak is likely to have been not maintaining the cold chain after food preparation. Whole genome sequencing resulted in phylogenetic clustering which concurred with traditional typing while simultaneously characterising virulence and resistance traits.

Short communication: Staphylococcus aureus isolated from colostrum of dairy heifers represent a closely related group exhibiting highly homogeneous genomic and antimicrobial resistance features.

In heifers, intramammary infections caused by Staphylococcus aureus affect milk production and udder health in the first and subsequent lactations, and can lead to premature culling. Not much is known about Staph. aureus isolated from heifers and it is also unclear whether or not these strains are readily transmitted between heifers and lactating herd mates. In this study, we compared phenotypic characteristics, spa types, and DNA microarray virulence and resistance gene profiles of Staph. aureus isolates obtained from colostrum samples of dairy heifers with isolates obtained from lactating cows. Our objective was to (1) characterize Staph. aureus strains associated with mastitis in heifers and (2) determine relatedness of Staph. aureus strains from heifers and lactating cows to provide data on transmission. We analyzed colostrum samples of 501 heifers and milk samples of 68 lactating cows within the same herd, isolating 48 and 9 Staph. aureus isolates, respectively. Staphylococcus aureus strains from heifers, lactating herd mates, and an unrelated collection of 78 strains from bovine mastitis milk of mature cows were compared. With 1 exception each, characterization of all strains from heifers and lactating cows in the same herd yielded highly similar phenotypic and genotypic results. The strains were Staphaurex latex agglutination test negative (Oxoid AG, Basel, Switzerland) and belonged to agr type II, CC705, and spa types tbl 2645 and t12926. They were susceptible to all antimicrobial agents tested. In contrast, the strains from mature cows in other herds were spread across different clonal complexes, spa types, and SplitsTree clusters (http://www.splitstree.org/), thus displaying a far higher degree of heterogeneity. We conclude that strains isolated from colostrum of heifers and mastitis milk of lactating cows in the same herd feature highly similar phenotypic and genomic characteristics, suggesting persistence of the organism during the first and potentially subsequent lactations or transmission between heifers and mature herd mates.

Outbreak of Staphylococcal food poisoning due to SEA-producing Staphylococcus aureus.

In 2008, 150 people gathered for a wedding celebration in Baden-Württemberg, Germany. Three hours after ingestion of a variety of foods including pancakes filled with minced chicken, several guests exhibited symptoms of acute gastroenteritis such as vomiting, diarrhea, fever, and ague. Twelve guests were reported to have fallen ill, with nine of these seeking medical care in hospitals. At least four patients were admitted to the hospital and received inpatient treatment, among them a 2-year-old child and a woman in the 4th month of pregnancy. Within 24 h of the event, an investigative team collected a variety of samples including refrigerated leftovers, food in the storage unit of the caterer, nasal swabs of the caterer, as well as 21 environmental swabs. Five stool samples from patients were provided by the hospitals. Staphylococcus aureus isolates were gathered from eight samples, among them nasal swabs of the caterer, food samples, and one stool sample. Fourier transform-infrared spectroscopy was used for species identification and for primary clustering of the isolates in a similarity tree. The isolates were further characterized by spa typing and pulsed-field gel electrophoresis, and a DNA microarray was used to determine the presence/absence of genes involved in virulence and antimicrobial resistance. We were able to match an enterotoxigenic strain from the stool sample of a patient to isolates of the same strain obtained from food and the nasal cavity of a food handler. The strain produced the enterotoxin SEA and the toxic shock syndrome toxin-1, and was also found to exhibit the genes encoding enterotoxins SEG and SEI, as well as the enterotoxin gene cluster egc. This is one of only a few studies that were able to link a staphylococcal food poisoning outbreak to its source.

Stress Lowers Staphylococcal Enterotoxin C Production Independently of Agr, SarA, and SigB.

Staphylococcal enterotoxin C (SEC) can cause staphylococcal food poisoning, one of the most prevalent foodborne intoxications. It is produced by Staphylococcus aureus during growth in the food matrix. While the surrounding bacteria in food matrices usually repress the growth of S.aureus, the organism possesses a remarkable growth advantage under stressful conditions encountered in many foods. Examples for such food matrices are pastry and bakery products with their high sugar content that lowers water availability. While S. aureus can still grow in these challenging environments, it remains unclear how these conditions affect SEC expression. Here, the influence of 30% glucose on sec mRNA in a qPCR assay and SEC protein expression was investigated for the first time in an ELISA. In addition, regulatory knockout mutants Δagr, ΔsarA, and ΔsigB were generated to investigate regulatory gene elements in glucose stress. In five out of seven strains, glucose stress led to a pronounced decrease in sec mRNA transcription and SEC protein levels were substantially lower under glucose stress. It could be shown that key regulatory elements Δagr, ΔsarA, and ΔsigB in strain SAI48 did not contribute to the pronounced downregulation under glucose stress. Based on these findings, glucose effectively lowers SEC synthesis in the food matrix. However, the mechanism by which it acts on toxin expression and regulatory elements in S. aureus remains unclear. Future studies on other regulatory elements and transcriptomics may shed light on the mechanisms.

PorinPredict: In Silico Identification of OprD Loss from WGS Data for Improved Genotype-Phenotype Predictions of P. aeruginosa Carbapenem Resistance.

The increasing integration of genomics into routine clinical diagnostics requires reliable computational tools to identify determinants of antimicrobial resistance (AMR) from whole-genome sequencing data. Here, we developed PorinPredict, a bioinformatic tool that predicts defects of the Pseudomonas aeruginosa outer membrane porin OprD, which are strongly associated with reduced carbapenem susceptibility. PorinPredict relies on a database of intact OprD variants and reports inactivating mutations in the coding or promoter region. PorinPredict was validated against 987 carbapenemase-negative P. aeruginosa genomes, of which OprD loss was predicted for 454 out of 522 (87.0%) meropenem-nonsusceptible and 46 out of 465 (9.9%) meropenem-susceptible isolates. OprD loss was also found to be common among carbapenemase-producing isolates, resulting in even further increased MICs. Chromosomal mutations in quinolone resistance-determining regions and OprD loss commonly co-occurred, likely reflecting the restricted use of carbapenems for multidrug-resistant infections as recommended in antimicrobial stewardship programs. In combination with available AMR gene detection tools, PorinPredict provides a robust and standardized approach to link P. aeruginosa phenotypes to genotypes. IMPORTANCE Pseudomonas aeruginosa is a major cause of multidrug-resistant nosocomial infections. The emergence and spread of clones exhibiting resistance to carbapenems, a class of critical last-line antibiotics, is therefore closely monitored. Carbapenem resistance is frequently mediated by chromosomal mutations that lead to a defective outer membrane porin OprD. Here, we determined the genetic diversity of OprD variants across the P. aeruginosa population and developed PorinPredict, a bioinformatic tool that enables the prediction of OprD loss from whole-genome sequencing data. We show a high correlation between predicted OprD loss and meropenem nonsusceptibility irrespective of the presence of carbapenemases, which are a second widespread determinant of carbapenem resistance. Isolates with resistance determinants to other antibiotics were disproportionally affected by OprD loss, possibly due to an increased exposure to carbapenems. Integration of PorinPredict into genomic surveillance platforms will facilitate a better understanding of the clinical impact of OprD modifications and transmission dynamics of resistant clones.

Mild NaCl Stress Influences Staphylococcal Enterotoxin C Transcription in a Time-Dependent Manner and Reduces Protein Expression.

Enterotoxins (SEs) produced by Staphylococcus aureus are the cause of serious food intoxications. Staphylococcal enterotoxin C (SEC) is one of the main contributors, as it is often highly expressed. S. aureus possesses a competitive growth advantage over accompanying bacterial flora under stress conditions encountered in foods, such as high NaCl concentrations. However, the influence of NaCl as an external stressor on SEC expression is still unclear. We investigated the influence of 4.5% NaCl on sec mRNA and SEC protein levels. A qRT-PCR assay revealed that NaCl stress leads to time-dependently decreased or elevated sec mRNA levels for most strains. SEC protein levels were generally decreased under NaCl stress. Our findings suggest that NaCl stress lowers overall SEC concentration and time-dependently affects sec mRNA levels.

Ropiness in Bread-A Re-Emerging Spoilage Phenomenon.

As bread is a very important staple food, its spoilage threatens global food security. Ropy bread spoilage manifests in sticky and stringy degradation of the crumb, slime formation, discoloration, and an odor reminiscent of rotting fruit. Increasing consumer demand for preservative-free products and global warming may increase the occurrence of ropy spoilage. Bacillus amyloliquefaciens, B. subtilis, B. licheniformis, the B. cereus group, B. pumilus, B. sonorensis, Cytobacillus firmus, Niallia circulans, Paenibacillus polymyxa, and Priestia megaterium were reported to cause ropiness in bread. Process hygiene does not prevent ropy spoilage, as contamination of flour with these Bacillus species is unavoidable due to their occurrence as a part of the endophytic commensal microbiota of wheat and the formation of heat-stable endospores that are not inactivated during processing, baking, or storage. To date, the underlying mechanisms behind ropy bread spoilage remain unclear, high-throughput screening tools to identify rope-forming bacteria are missing, and only a limited number of strategies to reduce rope spoilage were described. This review provides a current overview on (i) routes of entry of Bacillus endospores into bread, (ii) bacterial species implicated in rope spoilage, (iii) factors influencing rope development, and (iv) methods used to assess bacterial rope-forming potential. Finally, we pinpoint key gaps in knowledge and related challenges, as well as future research questions.

Nitrite stress increases staphylococcal enterotoxin C transcription and triggers the SigB regulon.

Staphylococcal food poisoning is a common food intoxication caused by staphylococcal enterotoxins. While growth of Staphylococcus aureus is not inhibited by the meat-curing agent nitrite, we hypothesize that nitrite has an influence on enterotoxin C (SEC) expression. We investigated the influence of 150 mg/l nitrite on SEC expression at mRNA and protein level in seven strains expressing different SEC variants. Additionally, regulatory knockout mutants (Δagr, ΔsarA, and ΔsigB) of high SEC producing strain SAI48 were investigated at mRNA level. Our findings suggest that nitrite effectively increases sec mRNA transcription, but the effects on SEC protein expression are less pronounced. While Δagr mutants exhibited lower sec mRNA transcription levels than wildtype strains, this response was not stress specific. ΔsigB mutants displayed a nitrite stress-specific response. Whole genome sequencing of the strains revealed a defective agr element in one strain (SAI3). In this strain, sec transcription and SEC protein synthesis was not affected by the mutation. Consequently, additional regulatory networks must be at play in SEC expression. Comparison of our findings about SEC with previous experiments on SEB and SED suggest that each SE can respond differently, and that the same stressor can trigger opposing responses in strains that express multiple toxins.

Mild Lactic Acid Stress Causes Strain-Dependent Reduction in SEC Protein Levels.

Staphylococcal enterotoxin C (SEC) is a major cause of staphylococcal food poisoning in humans and plays a role in bovine mastitis. Staphylococcus aureus (S. aureus) benefits from a competitive growth advantage under stress conditions encountered in foods such as a low pH. Therefore, understanding the role of stressors such as lactic acid on SEC production is of pivotal relevance to food safety. However, stress-dependent cues and their effects on enterotoxin expression are still poorly understood. In this study, we used human and animal strains harboring different SEC variants in order to evaluate the influence of mild lactic acid stress (pH 6.0) on SEC expression both on transcriptional and translational level. Although only a modest decrease in sec mRNA levels was observed under lactic acid stress, protein levels showed a significant decrease in SEC levels for some strains. These findings indicate that post-transcriptional modifications can act in SEC expression under lactic acid stress.

Further Insights into the Toxicity of Bacillus cytotoxicus Based on Toxin Gene Profiling and Vero Cell Cytotoxicity Assays.

Bacillus cytotoxicus belongs to the Bacillus cereus group that also comprises the foodborne pathogen Bacillus cereus sensu stricto, Bacillus anthracis causing anthrax, as well as the biopesticide Bacillus thuringiensis. The first B. cytotoxicus was isolated in the context of a severe food poisoning outbreak leading to fatal cases of diarrheal disease. Subsequent characterization of the outbreak strain led to the conclusion that this Bacillus strain was highly cytotoxic and eventually resulted in the description of a novel species, whose name reflects the observed toxicity: B. cytotoxicus. However, only a few isolates of this species have been characterized with regard to their cytotoxic potential and the role of B. cytotoxicus as a causative agent of food poisoning remains largely unclear. Hence, the aim of this study was to gain further insights into the toxicity of B. cytotoxicus. To this end, 19 isolates were obtained from mashed potato powders and characterized by toxin gene profiling and Vero cell cytotoxicity assays. All isolates harbored the cytK1 (cytotoxin K1) gene and species-specific variants of the nhe (non-hemolytic enterotoxin) gene. The isolates exhibited low or no toxicity towards Vero cells. Thus, this study indicates that the cytotoxic potential of B. cytotoxicus may be potentially lower than initially assumed.

Whole Genome Sequencing Reveals Biopesticidal Origin of Bacillus thuringiensis in Foods.

Bacillus thuringiensis is a microbial insecticide widely used to control agricultural pests. Although generally regarded as safe, B. thuringiensis is phylogenetically intermingled with the foodborne pathogen B. cereus sensu stricto and has been linked to foodborne outbreaks. Limited data on the pathogenicity potential of B. thuringiensis and the occurrence of biopesticide residues in food compromise a robust consumer risk assessment. In this study, we analyzed whole-genome sequences of 33 B. thuringiensis isolates from biopesticides, food, and human fecal samples linked to outbreaks. All food and outbreak-associated isolates genomically matched (≤ 6 wgSNPs; ≤ 2 cgSNPs) with one of six biopesticide strains, suggesting biopesticide products as their source. Long-read sequencing revealed a more diverse virulence gene profile than previously assumed, including a transposase-mediated disruption of the promoter region of the non-hemolytic enterotoxin gene nhe and a bacteriophage-mediated disruption of the sphingomyelinase gene sph in some biopesticide strains. Furthermore, we provide high-quality genome assemblies of seven widely used B. thuringiensis biopesticide strains, which will facilitate improved microbial source tracking and risk assessment of B. thuringiensis-based biopesticides in the future.

Genes involved in yellow pigmentation of Cronobacter sakazakii ES5 and influence of pigmentation on persistence and growth under environmental stress.

Cronobacter spp. are opportunistic food-borne pathogens that are responsible for rare but highly fatal cases of meningitis and necrotizing enterocolitis in neonates. While the operon responsible for yellow pigmentation in Cronobacter sakazakii strain ES5 was described recently, the involvement of additional genes in pigment expression and the influence of pigmentation on the fitness of Cronobacter spp. have not been investigated. Thus, the aim of this study was to identify further genes involved in pigment expression in Cronobacter sakazakii ES5 and to assess the influence of pigmentation on growth and persistence under conditions of environmental stress. A knockout library was created using random transposon mutagenesis. The screening of 9,500 mutants for decreased pigment production identified 30 colorless mutants. The mapping of transposon insertion sites revealed insertions in not only the carotenoid operon but also in various other genes involved in signal transduction, inorganic ions, and energy metabolism. To determine the effect of pigmentation on fitness, colorless mutants (DeltacrtE, DeltacrtX, and DeltacrtY) were compared to the yellow wild type using growth and inactivation experiments, a macrophage assay, and a phenotype array. Among other findings, the colorless mutants grew at significantly increased rates under osmotic stress compared to that of the yellow wild type while showing increased susceptibility to desiccation. Moreover, DeltacrtE and DeltacrtY exhibited increased sensitivity to UVB irradiation.