Bacillus Cereus Group Research Articles

Genes under positive selection in the core genome of pathogenic Bacillus cereus group members

In this comparative genomics study our aim was to unravel genes under positive selection in the core genome of the Bacillus cereus group. Indeed, the members of this group share close genetic relationships but display a rather large phenotypic and ecological diversity, providing a unique opportunity for studying how genomic changes reflect ecological adaptation during the divergence of a bacterial group. For this purpose, we screened ten completely sequenced genomes of four pathogenic Bacillus species, finding that 254 out of 3093 genes have codon sites with dN/dS (ω) values above one. These results remained unchanged after having disentangled the confounding effects of recombination and selection signature in a Bayesian framework. The presumably adaptive nucleotide polymorphisms are distributed over a wide range of biological functions, such as antibiotic resistance, DNA repair, nutrient uptake, metabolism, cell wall assembly and spore structure. Our results indicate that adaptation to animal hosts, whether as pathogens, saprophytes or symbionts, is the major driving force in the evolution of the Bacillus cereus group. Future work should seek to understand the evolutionary dynamics of both core and accessory genes in an integrative framework to ultimately unravel the key networks involved in host adaptation.

Edible processed insects from e-commerce: Food safety with a focus on the Bacillus cereus group

Edible insects are increasing in popularity as a trendy new food item in many countries worldwide. However, little information is available regarding the associated foodborne pathogens, in particular, the spore-forming bacteria that may be present in these processed foods. In this study, mealworms, crickets, mole crickets and silkworms that are widely available online were investigated for microbial quality traits.Whereas water activity and pH results revealed a stable product, microbiota characterisation highlighted wide variability among the analysed insect species. Among the microbial targets considered in this study, total viable count, total aerobic spores and Bacillus cereus count were the most suitable for describing the food safety and microbial quality of these products. Endospore-forming bacteria ascribed to the B. cereus group were identified with genetic methods and putative virulence patterns. Three major clusters were delineated according to the phylogenetic analysis of the B. cereus group: B. cereus, B. thuringiensis and B. cytotoxicus, with virulence patterns particular to the enterotoxin genes found in each cluster.This work represents a first step in the hazard identification of B. cereus group bacteria isolated from edible insects. The presented results should also be considered with respect to domestic handling and rehydration of such products prior to consumption.

Endogenous microbial contamination of melons (Cucumis melo) from international trade: an underestimated risk for the consumer?

Fruits and vegetables have increasingly been related to foodborne outbreaks. Besides surface contamination, a possible internalization of microorganisms into edible parts of plants during growth has already been observed. To examine an actual risk for the consumer, microbial contamination of the rind and pulp of 147 muskmelons from international trade was assessed using cultural and biochemical methods, polymerase chain reaction and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. One hundred percent of the rind samples [3.69-8.92 log colony forming units (CFU) g-1 ] and 89.8% of the pulp samples (maximum load 3.66 log CFU g-1 ) were microbiologically contaminated. Among the 432 pulp isolates, opportunistic and potentially pathogenic bacteria were identified, mainly Staphylococcus spp. (48.9%), Clostridium spp. (42.9%) and Enterobacteriaceae (27.9%). Salmonella spp., Escherichia coli and isolates of the Bacillus cereus group were found on the rind (1.4%, 0.7% and 42.9%, respectively) and in the pulp (0.7%, 1.4% and 4.7%). Clostridium perfringens was isolated from the rind of seven melons. The present study revealed a regularly occurring internal contamination of melons. Possible health risks for consumers because of an occurrence of microorganisms in melon pulp should be considered in future food safety assessments. © 2018 Society of Chemical Industry.

Corrigendum: Bacteriophages and bacteriophage-derived endolysins as potential therapeutics to combat Gram-positive sporeforming bacteria.

Since their discovery in 1915, bacteriophages have been routinely used within Eastern Europe to treat a variety of bacterial infections. Although initially ignored by the West due to the success of antibiotics, increasing levels and diversity of antibiotic resistance is driving a renaissance for bacteriophage-derived therapy, which is in part due to the highly specific nature of bacteriophages as well as their relative abundance. This review focuses on the bacteriophages and derived lysins of relevant Gram-positive spore formers within the Bacillus cereus group and Clostridium genus that could have applications within the medical, food and environmental sectors.

Lineage-specific plasmid acquisition and the evolution of specialized pathogens in Bacillus thuringiensis and the Bacillus cereus group.

Bacterial plasmids can vary from small selfish genetic elements to large autonomous replicons that constitute a significant proportion of total cellular DNA. By conferring novel function to the cell, plasmids may facilitate evolution but their mobility may be opposed by co‐evolutionary relationships with chromosomes or encouraged via the infectious sharing of genes encoding public goods. Here, we explore these hypotheses through large‐scale examination of the association between plasmids and chromosomal DNA in the phenotypically diverse Bacillus cereus group. This complex group is rich in plasmids, many of which encode essential virulence factors (Cry toxins) that are known public goods. We characterized population genomic structure, gene content and plasmid distribution to investigate the role of mobile elements in diversification. We analysed coding sequence within the core and accessory genome of 190 B. cereus group isolates, including 23 novel sequences and genes from 410 reference plasmid genomes. While cry genes were widely distributed, those with invertebrate toxicity were predominantly associated with one sequence cluster (clade 2) and phenotypically defined Bacillus thuringiensis. Cry toxin plasmids in clade 2 showed evidence of recent horizontal transfer and variable gene content, a pattern of plasmid segregation consistent with transfer during infectious cooperation. Nevertheless, comparison between clades suggests that co‐evolutionary interactions may drive association between plasmids and chromosomes and limit wider transfer of key virulence traits. Proliferation of successful plasmid and chromosome combinations is a feature of specialized pathogens with characteristic niches (Bacillus anthracis, B. thuringiensis) and has occurred multiple times in the B. cereus group.

Microbial diversity and activity of an aged soil contaminated by polycyclic aromatic hydrocarbons.

In-depth understanding of indigenous microbial assemblages resulted from aged contamination by polycyclic aromatic hydrocarbons (PAHs) is of vital importance in successful in situ bioremediation treatments. Soil samples of three boreholes were collected at 12 different vertical depths. Overall, the dominating three-ring PAHs (76.2%) were closely related to distribution patterns of soil dehydrogenase activity, microbial cell numbers, and Shannon biodiversity index (H') of indigenous microorganisms. High-molecular-weight PAHs tend to yield more diverse communities. Results from 16S rRNA analysis indicated that possible functional groups of PAH degradation include three species in Bacillus cereus group, Bacillus sp. SA Ant14, Nocardioides sp., and Ralstonia pickettii. Principal component analysis indicates significant positive correlations between the content of high-molecular-weight PAHs and the distribution of Bacillus weihenstephanensis KBAB4 and Nocardioides sp. The species B. cereus 03BB102, Bacillus thuringiensis, B. weihenstephanensis KBAB4, and Nocardioides sp. were recognized as main PAH degraders and thus recommended to be utilized in further bioremediation applications. The vertical distribution characteristics of PAHs in soil profiles (1-12m) and the internal relationship between the transport mechanisms of PAHs and the response of soil biological properties help further understand the microbial diversity and activity in an aged site.

Isolation, Identification, Prevalence, and Genetic Diversity of Bacillus cereus Group Bacteria From Different Foodstuffs in Tunisia.

Bacillus cereus group is widespread in nature and foods. Several members of this group are recognized as causing food spoilage and/or health issues. This study was designed to determine the prevalence and genetic diversity of the B. cereus group strains isolated in Tunisia from different foods (cereals, spices, cooked food, fresh-cut vegetables, raw and cooked poultry meats, seafood, canned, pastry, and dairy products). In total, 687 different samples were collected and searched for the presence of the B. cereus group after selective plating on MYP agar and enumeration of each sample. The typical pink-orange uniform colonies surrounded by a zone of precipitate were assumed to belong to the B. cereus group. One typical colony from each sample was subcultured and preserved as cryoculture. Overall, 191 (27.8%) food samples were found positive, giving rise to a collection of 191 B. cereus-like isolates. The concentration of B. cereus-like bacteria were below 103 cfu/g or ml in 77.5% of the tested samples. Higher counts (>104 cfu/g or ml) were found in 6.8% of samples including fresh-cut vegetables, cooked foods, cereals, and pastry products. To verify whether B. cereus-like isolates belonged to the B. cereus group, a PCR test targeting the sspE gene sequence specific of the group was carried out. Therefore, 174 isolates were found to be positive. Food samples were contaminated as follows: cereals (67.6%), pastry products (46.2%), cooked food (40.8%), cooked poultry meat (32.7%), seafood products (32.3%), spices (28.8%), canned products (16.7%), raw poultry meat (9.4%), fresh-cut vegetables (5.0%), and dairy products (4.8%). The 174 B. cereus isolates were characterized by partial sequencing of the panC gene, using a Sym'Previous software tool to assign them to different phylogenetic groups. Strains were distributed as follows: 61.3, 29.5, 7.5, and 1.7% in the group III, IV, II, and V, respectively. The genetic diversity was further assessed by ERIC-PCR and PFGE typing methods. PFGE and ERIC-PCR patterns analysis allowed discriminating 143 and 99 different profiles, respectivey. These findings, associated to a relatively higher prevalence of B. cereus group in different foods, could be a significant etiological agent of food in Tunisia.

Application of a novel amplicon-based sequencing approach reveals the diversity of the Bacillus cereus group in stored raw and pasteurized milk

Members of the Bacillus cereus sensu lato (B. cereus group) are spore-forming organisms commonly associated with spoilage of milk and dairy products. Previous studies have shown, by using 16S marker gene sequencing, that the genus Bacillus is part of the core microbiota of raw bovine milk and that some members of this genus are able to grow during sub-optimal storage (8 °C) of pasteurized consumption milk. Here, the composition of this genus in pasteurized consumption milk samples, collected from two dairies, over a one-year period and stored at 4 or 8 °C up to the end of shelf life is uncovered. Our results show that the B. cereus group is the dominant Bacillus group in stored consumption milk. By applying a new marker gene sequencing approach, several dominating phylogenetic clusters were identified within the B. cereus group populations from the milk samples. There was a higher phylogenetic diversity among bacteria from milk stored at 8 °C compared to milk stored at 4 °C. Sampling period and the dairy the samples were collected from, also significantly influenced the diversity, which shows that the B. cereus group population in consumption milk is heterogeneous and subjected to temporal and spatial changes. The new approach applied in this study will facilitate the identification of isolates within the B. cereus group, of which some are potential spoilage bacteria and pathogenic contaminants of milk and dairy products.

Diversity and enzymatic potentialities of Bacillus sp. strains isolated from a polluted freshwater ecosystem in Cuba.

Genotypic and phenotypic characterization of Bacillus spp. from polluted freshwater has been poorly addressed. The objective of this research was to determine the diversity and enzymatic potentialities of Bacillus spp. strains isolated from the Almendares River. Bacilli strains from a polluted river were characterized by considering the production of extracellular enzymes using API ZYM. 14 strains were selected and identified using 16S rRNA, gyrB and aroE genes. Genotypic diversity of the Bacillus spp. strains was evaluated using pulsed field gel electrophoresis. Furthermore, the presence of genetic determinants of potential virulence toxins of the Bacillus cereus group and proteinaceous crystal inclusions of Bacillus thuringiensis was determined. 10 strains were identified as B. thuringiensis, two as Bacillus megaterium, one as Bacillus pumilus and one as Bacillus subtilis. Most strains produced proteases, amylases, phosphatases, esterases, aminopeptidases and glucanases, which reflect the abundance of biopolymeric matter in Almendares River. Comparison of the typing results revealed a spatio-temporal distribution among B. thuringiensis strains along the river. The results of the present study highlight the genotypic and phenotypic diversity of Bacillus spp. strains from a polluted river, which contributes to the knowledge of genetic diversity of Bacilli from tropical polluted freshwater ecosystems.

Molecular discrimination of Bacillus cereus group species in foods (lettuce, spinach, and kimbap) using quantitative real-time PCR targeting groEL and gyrB

The aim of the study was to identify and evaluate specific biomarkers to differentiate within Bacillus cereus group species from contaminated food samples with the use of real-time PCR. A total of 120 strains, comprising of 28 reference, 2 type, 78 wild strains of B. cereus and B. thuringiensis along with 12 strains representing 2 bacterial groups – B. mycoides, B. pseudomycoides, B. weihenstephanensis (B. cereus group); B. amyloliquefaciens, B. subtilis, Enterococcus faecalis, Escherichia coli, Listeria monocytogenes, Micrococcus luteus, Salmonella enterica, Staphylococcus aureus, Streptococcus pyogenes (non-Bacillus sp.) were identified by applying valid biomarkers (groEL and gyrB). In addition, the presence of B. cereus group was determined in three different artificially contaminated vegetable samples (lettuce, spinach, and kimbap), using prominent biomarkers targeting on chaperonin protein (GroEL) and topoisomerase enzyme protein (gyrB). Direct analysis of samples revealed the specificity towards identification and characterization of the B. cereus group among wild, reference and type strains and the type strain inoculated in vegetables. Our results demonstrated two existing biomarkers groEL and gyrB with a high specificity of 98% and 96% respectively to analyze the total B. cereus group. Further, we also reported the detection limit of groEL and gyrB in food samples was 3.5 and 3.7 log CFU/g respectively. Thus, the developed real-time PCR approach can be a reliable and effective tool for the identification of B. cereus group strains present in environment and food samples. This does not require band isolation, re-amplification, sequencing or sequence identification, thus reducing the time and cost of analysis.

A minireplicon of plasmid pBMB26 represents a new typical replicon in the megaplasmids of Bacillus cereus group.

A new minireplicon (rep26 minireplicon) from pBMB26, the 188 kb indigenous plasmid related to spore-crystal association (SCA) phenotype in Bacillus thuringiensis strain YBT-020, was characterized. A 12 kb EcoRI fragment, which encoded 10 putative open reading frames (ORFs), was capable of supporting replication when cloned in a replication probe vector. Deletion and frame shift mutation analysis showed that a 4.1 kb region encompassing two putative ORFs (orf21 and orf22) was essential for the plasmid replication in B. thuringiensis. Gene orf21 encoding a 49.8 kDa protein (named Rep26) with a helix-turn-helix motif showed no homology with known replication proteins and gene orf22 encoding a protein of 82.6 kDa showed homology to bacterial PcrA helicase. The replication origin of rep26 minireplicon was proved to be located in the coding region of orf21. Plasmid stability experiments indicated that the recombinant plasmid containing rep26 minireplicon has excellent segregational stability. BLASTP analysis revealed that amino acid sequences of ORF21 and ORF22 were well conserved among Bacillus cereus group strains. The rep26 minireplicon was widely distributed and could be defined as a new typical replicon in the megaplasmids of B. cereus group.

RNA-Seq Analysis of Antibiotic-Producing Bacillus subtilis SC-8 Reveals a Role for Small Peptides in Controlling PapR Signaling.

Bacillus subtilis SC-8 (BSSC8) shows a narrow antimicrobial activity against the Bacillus cereus group. Previously, B. cereus-derived PapR as a signal peptide to stimulate PlcR, which plays a significant role in regulating the transcription of virulence factors, was assumed to stimulate antibiotic production in BSSC8. To better understand the functional role of PapR in the antibiotic production of BSSC8 and the interspecies interaction, the global transcriptomic profiling of BSSC8 was investigated using RNA-Seq in this study. Small peptides derived from B. cereus wild type (WTBC) and a papR-deleted mutant strain (MTBC) were individually supplied to BSSC8 cultures, and changes in global transcription levels were compared by RNA-Seq. In the presence of WTBC small peptides, more genes (80.9%) were significantly upregulated than in cells exposed to MTBC small peptides. Specifically, 48.8 and 83.4% of genes involved in glycolysis and the TCA cycle, respectively, showed changes in transcription levels in response to small peptides from both strains. Of the genes showing the alterations, 35.0% (glycolysis) and 60.0% (TCA cycle) of transcripts were significantly regulated only in response to WTBC-derived small peptides. Furthermore, the expression of biosynthetic genes encoding several known antibiotics in BSSC8 was further decreased in response to WTBC small peptides.

Bacillus Classification Based on Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry\u2014Effects of Culture Conditions

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a reliable and rapid technique applied widely in the identification and classification of microbes. MALDI-TOF MS has been used to identify many endospore-forming Bacillus species; however, endospores affect the identification accuracy when using MALDI-TOF MS because they change the protein composition of samples. Since culture conditions directly influence endospore formation and Bacillus growth, in this study we clarified how culture conditions influence the classification of Bacillus species by using MALDI-TOF MS. We analyzed members of the Bacillus subtilis group and Bacillus cereus group using different incubation periods, temperatures and media. Incubation period was found to affect mass spectra due to endospores which were observed mixing with vegetative cells after 24 hours. Culture temperature also resulted in different mass spectra profiles depending on the temperature best suited growth and sporulation. Conversely, the four common media for Bacillus incubation, Luria-Bertani agar, nutrient agar, plate count agar and brain-heart infusion agar did not result in any significant differences in mass spectra profiles. Profiles in the range m/z 1000–3000 were found to provide additional data to the standard ribosomal peptide/protein region m/z 3000–15000 profiles to enable easier differentiation of some highly similar species and the identification of new strains under fresh culture conditions. In summary, control of culture conditions is vital for Bacillus identification and classification by MALDI-TOF MS.

Bacterial Whole Cell Typing by Mass Spectra Pattern Matching with Bootstrapping Assessment.

Bacterial typing is of great importance in clinical diagnosis, environmental monitoring, food safety analysis, and biological research. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is now widely used to analyze bacterial samples. Identification of bacteria at the species level can be realized by matching the mass spectra of samples against a library of mass spectra of known bacteria. Nevertheless, in order to reasonably type bacteria, identification accuracy should be further improved. Herein, we propose a new framework to the identification and assessment for MALDI-MS based bacterial analysis. Our approach combines new measures for spectra similarity and a novel bootstrapping assessment. We tested our approach on a general data set containing the mass spectra of 1741 strains of bacteria and another challenging data set containing 250 strains, including 40 strains in the Bacillus cereus group that were previously claimed to be impossible to resolve by MALDI-MS. With the bootstrapping assessment, we achieved much more reliable predictions at both the genus and species level, and enabled to resolve the Bacillus cereus group. To the best of the authors' knowledge, our method is the first to provide a statistical assessment to MALDI-MS based bacterial typing that could lead to more reliable bacterial typing.

PatB1 is an O-acetyltransferase that decorates secondary cell wall polysaccharides.

O-Acetylation of the secondary cell wall polysaccharides (SCWP) of the Bacillus cereus group of pathogens, which includes Bacillus anthracis, is essential for the proper attachment of surface-layer (S-layer) proteins to their cell walls. Using a variety of pseudosubstrates and a chemically synthesized analog of SCWP, we report here the identification of PatB1 as a SCWP O-acetyltransferase in Bacillus cereus. Additionally, we report the crystal structure of PatB1, which provides detailed insights into the mechanism of this enzyme and defines a novel subfamily of the SGNH family of esterases and lipases. We propose a model for the O-acetylation of SCWP requiring the translocation of acetyl groups from a cytoplasmic source across the plasma membrane by PatA1 and PatA2 for their transfer to SCWP by PatB1.

Genomic Analysis of Bacillus sp. Strain B25, a Biocontrol Agent of Maize Pathogen Fusarium verticillioides

Bacillus sp. B25 is an effective biocontrol agent against the maize pathogenic fungus Fusarium verticillioides (Fv). Previous in vitro assays have shown that B25 has protease, glucanase, and chitinase activities and siderophores production; however, specific mechanisms by which B25 controls Fv are still unknown. To determine the genetic traits involved in biocontrol, B25 genome was sequenced and analyzed. B25 genome is composed of 5,113,413bp and 5251 coding genes. A multilocus phylogenetic analysis (MLPA) suggests that B25 is closely related to the Bacillus cereus group and a high percentage (70-75%) of the genetic information is conserved between B25 and related strains, which include most of the genes associated to fungal antagonism. Some of these genes are shared with some biocontrol agents of the Bacillus genus and less with Pseudomonas and Serratia strains. We performed a genomic comparison between B25 and five Bacillus spp., Pseudomonas and Serratia strains. B25 contains genes involved in a wide variety of antagonistic mechanisms including chitinases, glycoside hydrolases, siderophores, antibiotics, and biofilm production that could be implicated in root colonization. Also, 24 genomic islands and 3 CRISPR sequences were identified in the B25 genome. This is the first comparative genome analysis between strains belonging to the B. cereus group and biocontrol agents of phytopathogenic fungi. These results are the starting point for further studies on B25 gene expression during its interaction with Fv.

Toxin production ability of Bacillus cereus strains from food product of Ukraine

Potential pathogens of foodborne toxic infections – bacterial contaminants Bacillus cereus isolated from plant raw materials and food products from the Ukrainian region were investigated. When determining of the proportion of isolated bacilli from the plant samples, it was established that the epidemiologically significant microorganisms of Bacillus cereus as agents of food poisoning are the second largest. The average value of contaminated samples of Ukrainian plant raw materials and processed products with Bacillus cereus is 36,2 %. The ability of Bacillus cereus strains identified by a complex of morphological, tinctorial, cultural and biochemical properties, to produce specific emetic and enterotoxins was studied. Molecular genetic diagnosis and detection of the toxin-producing ability of isolated 42 Bacillus cereus strains showed both the possibility of their rapid identification and the presence of specific toxicity genes. Multiplex polymerase chain reaction (PCR) was carried out with specific primers to detect toxicity determined of various bacilli genes: nheA, hblD, cytK, cesВ. The distribution of toxigenic genes is significantly different among the Bacillus cereus isolates from various sources. The nheA, hblD and cytK enterotoxin genes were detected in 100, 83,3 and 61,9 % of the investigated strains of Bacillus cereus, respectively. The cesB gene encoding emetic toxin was detected in 4,8 % of strains. Molecular-genetic PCR-method confirmed that all the isolated strains belong to the Bacillus cereus group, and the ability to produce toxins can be attributed to five groups. The main toxins that produce the investigated Bacillus cereus strains were nhe and hbl enterotoxins encoded by the corresponding genes of nheA and hblD. The enterotoxic type of Bacillus cereus was predominant in Ukrainian region. Studies of domestic plant food raw materials and products have confirmed the need to improve microbiological control of product safety by introducing accelerated specific diagnostics of contaminants by molecular genetics methods.

Successful treatment ofBacillus cereusgroup brain abscesses with a combination of high-dose ciprofloxacin and vancomycin

Central nervous system (CNS) infections (e.g., brain abscess, meningitis) caused by members of the Bacillus cereus group are rare, but the associated crude mortality is significant. The optimal antimicrobial regimen for patients with these infections remains poorly defined. This report describes a case of multiple brain abscesses in an immunocompromised patient caused by a member of the B. cereus group. The patient was successfully treated with a combination of high-dose ciprofloxacin and vancomycin, providing evidence for the clinical utility of this therapeutic regimen.

Dynamics of culturable mesophilic bacterial communities of three fresh herbs and their production environment.

Investigate dynamics of culturable mesophilic bacteria and selected food-contaminating bacteria from three herbs and their production environment. Marjoram, basil and thyme were investigated during one growing season by sampling plants, organic fertilizers, soil, irrigation water and marketed products. Mesophilic bacteria and selected food-contaminating bacteria (Escherichia coli, Enterococcus spp., Bacillus cereus group) were cultured and identified by MALDI biotyping. Culturable mesophilic bacteria on marjoram and basil plants decreased over time by two orders of magnitude starting at above 106 colony forming units per gram (CFU per g), while they remained constant on thyme (~104 CFU per g). Compared to the last field sample, mesophilic bacteria were increased on all market-ready products by one order of magnitude. Marjoram and basil were dominated by B. cereus group, Enterobacter spp. and Pseudomonas spp., thyme by Bacillus spp. and Pseudomonas spp. All selected food-contaminating bacteria were detected in soil and reservoir-sourced irrigation water, whereas in municipal water, only B. cereus group and rarely Enterococcus spp. were found. Escherichia coli was detected only on young marjoram and basil plants (5×102 and 5×101 CFU per g, respectively), whereas Enterococcus spp. and B. cereus group were consistently detected on these two herbs. Thyme plants only contained B. cereus group consistently (above 103 CFU per g). Marketed marjoram and thyme contained Enterococcus spp. (5×102 and 104 CFU per g) and B. cereus group (~5×102 CFU per g), while no selected food-contaminating bacteria were found on marketed basil. Overall, culturable mesophilic bacteria were dominated by Pseudomonas spp. and Bacillus spp., with increased numbers on market-ready products. Selected food-contaminating bacteria were readily detectable, however, only the B. cereus group was found throughout in all systems. Insight into composition and development of mesophilic bacterial communities and selected food-contaminating bacteria of fresh herbs contributes to estimating consumer exposure.

Genetic Environment of cry1 Genes Indicates Their Common Origin.

Although in Bacillus thuringiensis the cry genes coding for the insecticidal crystal proteins are plasmid-borne and are usually associated with mobile genetic elements, several aspects related to their genomic organization, diversification, and transmission remain to be elucidated. Plasmids of B. thuringiensis and other members of the Bacillus cereus group (n = 364) deposited in GenBank were screened for the presence of cry1 genes, and their genetic environment was analyzed using a comparative bioinformatic approach. The cry1 genes were identified in 27 B. thuringiensis plasmids ranging from 64 to 761 kb, and were predominantly associated with the ori44, ori60, or double orf156/orf157 and pXO1-16/pXO1-14 replication systems. In general, the cry1 genes occur individually or as a part of an insecticidal pathogenicity island (PAI), and are preceded by genes coding for an N-acetylmuramoyl-l-alanine amidase and a putative K+(Na+)/H+ antiporter. However, except in the case of the PAI, the latter gene is disrupted by the insertion of IS231B. Similarly, numerous mobile elements were recognized in the region downstream of cry1, except for cry1I that follows cry1A in the PAI. Therefore, the cassette involving cry1 and these two genes, flanked by transposable elements, named as the cry1 cassette, was the smallest cry1-carrying genetic unit recognized in the plasmids. Conservation of the genomic environment of the cry1 genes carried by various plasmids strongly suggests a common origin, possibly from an insecticidal PAI carried by B. thuringiensis megaplasmids.