Characterization of Bacillus pumilus Strains Isolated from Bovine Uteri

Uterine infections are a major source of economic losses to dairy farmers. The uterine microbiota as well as opportunistic uterine contaminants can contribute to the development of endometritis in dairy cows during the postpartum period. Therefore, it is important to characterize potential pathogens and to further elucidate their role in the disease. In this study, we aimed to characterize Bacillus pumilus field isolates to obtain more details regarding their effect on uterine cells by using an in vitro endometrial epithelial primary cells model. We found that B. pumilus isolates possessed the keratinase genes ker1 and ker2 and therefore may produce keratinases. When primary endometrial epithelial cells were infected with 4 different B. pumilus strains, an effect on cellular viability was observed over the course of 72 h. The effect was dose-dependent and time-dependent. Nevertheless, significant differences between the strains were not observed. All tested strains reduced the viability of the primary cells after 72 h of incubation, indicating that B. pumilus potentially has a pathogenic effect on endometrial epithelial cells

Streptococcus uberis strains originating from bovine uteri provoke upregulation of pro-inflammatory factors mRNA expression of endometrial epithelial cells in vitro

Streptococcus uberis is an opportunistic pathogen involved in various infections of cattle. It is a well-known etiological agent of bovine mastitis and has recently also been linked to postpartum endometritis in dairy cows. S. uberis is frequently isolated from the uterus of postpartum cows but its actual contribution to host pathophysiology is unknown and information on S. uberis virulence factors potentially involved in the disease is lacking. To gain first insights into the role of S. uberis in the pathology of bovine endometritis, a cell-culture-based infection model was employed to study inflammatory host responses and investigate cytotoxic effects. A comprehensive strain panel, comprising 53 strains previously isolated from bovine uteri, was compiled and screened for known virulence factor genes. Isolates showing distinct virulence gene patterns were used to study their impact on cellular viability and influence on mRNA expression of pro-inflammatory factors in endometrial epithelial cells. Our study revealed that S. uberis negatively impacts the viability of endometrial epithelial cells and provokes an upregulation of specific pro-inflammatory factors, although with certain strains having a greater effect than others. Especially, mRNA expression of IL1A and CXCL8 as well as CXCL1/2 and PTGS2 was found to be stimulated by S. uberis. These results suggest that S. uberis might indeed contribute to the establishment of bovine endometritis

Proteome approaches combined with Fourier transform infrared spectroscopy revealed a distinctive biofilm physiology in Bordetella pertussis

Proteome analysis was combined with whole-cell metabolic fingerprinting to gain insight into the physiology of mature biofilm in Bordetella pertussis, the agent responsible for whooping cough. Recent reports indicate that B. pertussis adopts a sessile biofilm as a strategy to persistently colonize the human host. However, since research in the past mainly focused on the planktonic lifestyle of B. pertussis, knowledge on biofilm formation of this important human pathogen is still limited. Comparative studies were carried out by combining 2-DE and Fourier transform infrared (FT-IR) spectroscopy with multivariate statistical methods. These complementary approaches demonstrated that biofilm development has a distinctive impact on B. pertussis physiology. Results from MALDI-TOF/MS identification of proteins together with results from FT-IR spectroscopy revealed the biosynthesis of a putative acidic-type polysaccharide polymer as the most distinctive trait of B. pertussis life in a biofilm. Additionally, expression of proteins known to be involved in cellular regulatory circuits, cell attachment and virulence was altered in sessile cells, which strongly suggests a significant impact of biofilm development on B. pertussis pathogenesis. In summary, our work showed that the combination of proteomics and FT-IR spectroscopy with multivariate statistical analysis provides a powerful tool to gain further insight into bacterial lifestyles.Facultad de Ciencias ExactasCentro de Investigación y Desarrollo en Fermentaciones Industriale

Cereulide synthetase gene cluster from emetic Bacillus cereus: Structure and location on a mega virulence plasmid related to Bacillus anthracis toxin plasmid pXO1

BACKGROUND: Cereulide, a depsipeptide structurally related to valinomycin, is responsible for the emetic type of gastrointestinal disease caused by Bacillus cereus. Recently, it has been shown that this toxin is produced by a nonribosomal peptide synthetase (NRPS), but its exact genetic organization and biochemical synthesis is unknown. RESULTS: The complete sequence of the cereulide synthetase (ces) gene cluster, which encodes the enzymatic machinery required for the biosynthesis of cereulide, was dissected. The 24 kb ces gene cluster comprises 7 CDSs and includes, besides the typical NRPS genes like a phosphopantetheinyl transferase and two CDSs encoding enzyme modules for the activation and incorporation of monomers in the growing peptide chain, a CDS encoding a putative hydrolase in the upstream region and an ABC transporter in the downstream part. The enzyme modules responsible for incorporation of the hydroxyl acids showed an unusual structure while the modules responsible for the activation of the amino acids Ala and Val showed the typical domain organization of NRPS. The ces gene locus is flanked by genetic regions with high homology to virulence plasmids of B. cereus, Bacillus thuringiensis and Bacillus anthracis. PFGE and Southern hybridization showed that the ces genes are restricted to emetic B. cereus and indeed located on a 208 kb megaplasmid, which has high similarities to pXO1-like plasmids. CONCLUSION: The ces gene cluster that is located on a pXO1-like virulence plasmid represents, beside the insecticidal and the anthrax toxins, a third type of B. cereus group toxins encoded on megaplasmids. The ces genes are restricted to emetic toxin producers, but pXO1-like plasmids are also present in emetic-like strains. These data might indicate the presence of an ancient plasmid in B. cereus which has acquired different virulence genes over time. Due to the unusual structure of the hydroxyl acid incorporating enzyme modules of Ces, substantial biochemical efforts will be required to dissect the complete biochemical pathway of cereulide synthesis

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

Bovine Endometrial Epithelial Cells Scale Their Pro-inflammatory Response In vitro to Pathogenic Trueperella pyogenes Isolated from the Bovine Uterus in a Strain-Specific Manner

Among different bacteria colonizing the bovine uterus, Trueperella pyogenes is found to be associated with clinical endometritis (CE). The ability of cows to defend against T. pyogenes infections depends on the virulence of invading bacteria and on the host's innate immunity. Therefore, to gain insights into bacterial factors contributing to the interplay of this host pathogen, two strains of T. pyogenes were included in this study: one strain (TP2) was isolated from the uterus of a postpartum dairy cow developing CE and a second strain (TP5) was isolated from a uterus of a healthy cow. The two strains were compared in terms of their metabolic fingerprints, growth rate, virulence gene transcription, and effect on bovine endometrial epithelial cells in vitro. In addition, the effect of the presence of peripheral blood mononuclear cells (PBMCs) on the response of endometrial epithelial cells was evaluated. TP2, the strain isolated from the diseased cow, showed a higher growth rate, expressed more virulence factors (cbpA, nanH, fimE, and fimG), and elicited a higher mRNA expression of pro-inflammatory factors (PTGS2, CXCL3, and IL8) in bovine endometrial epithelial cells compared with TP5, the strain isolated from the healthy cow. The presence of PBMCs amplified the mRNA expression of pro-inflammatory factors (PTGS2, CXCL3, IL1A, IL6, and IL8) in bovine endometrial epithelial cells co-cultured with live TP2 compared with untreated cells, especially as early as after 4 h. In conclusion, particular strain characteristics of T. pyogenes were found to be important for the development of CE. Furthermore, immune cells attracted to the site of infection might also play an important role in up-regulation of the pro-inflammatory response in the bovine uterus and thus significantly contribute to the host-pathogen interaction

Rapid and reliable identification of Staphylococcus aureus capsular serotypes by means of artificial neural network-assisted Fourier-transform infrared spectroscopy

Staphylococcus aureus capsular polysaccharides (CP) are important virulence factors and represent putative targets for vaccine development. Therefore, the purpose of this study was to develop a high-throughput method to identify and discriminate the clinically important S. aureus capsular serotypes 5, 8, and NT (nontypeable). A comprehensive set of clinical isolates derived from different origins and control strains, representative for each serotype, were used to establish a CP typing system based on Fourier transform infrared (FTIR) spectroscopy and chemometric techniques. By combining FTIR spectroscopy with artificial neuronal network (ANN) analysis, a system was successfully established, allowing a rapid identification and discrimination of all three serotypes. The overall accuracy of the ANN-assisted FTIR spectroscopy CP typing system was 96.7% for the internal validation and 98.2% for the external validation. One isolate in the internal validation and one isolate in the external validation failed in the classification procedure, but none of the isolates was incorrectly classified. The present study demonstrates that ANN-assisted FTIR spectroscopy allows a rapid and reliable discrimination of S. aureus capsular serotypes. It is suitable for diagnostic as well as large-scale epidemiologic surveillance of S. aureus capsule expression and provides useful information with respect to chronicity of infection.Fil: Grunert, Tom. University of Veterinary Medicine; AustriaFil: Wenning, Mareike. Technische Universitat Munchen; AlemaniaFil: Barbagelata, María Sol. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Fricker, Martina. University of Veterinary Medicine; AustriaFil: Sordelli, Daniel Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Buzzola, Fernanda Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Ehling Schulz, Monika. University of Veterinary Medicine; Austri

Detection and Identification of Bacillus cereus, Bacillus cytotoxicus, Bacillus thuringiensis, Bacillus mycoides and Bacillus weihenstephanensis via Machine Learning Based FTIR Spectroscopy

The Bacillus cereus group comprises genetical closely related species with variable toxigenic characteristics. However, detection and differentiation of the B. cereus group species in routine diagnostics can be difficult, expensive and laborious since current species designation is linked to specific phenotypic characteristic or the presence of species-specific genes. Especially the differentiation of Bacillus cereus and Bacillus thuringiensis, the identification of psychrotolerant Bacillus mycoides and Bacillus weihenstephanensis, as well as the identification of emetic B. cereus and Bacillus cytotoxicus, which are both producing highly potent toxins, is of high importance in food microbiology. Thus, we investigated the use of a machine learning approach, based on artificial neural network (ANN) assisted Fourier transform infrared (FTIR) spectroscopy, for discrimination of B. cereus group members. The deep learning tool box of Matlab was employed to construct a one-level ANN, allowing the discrimination of the aforementioned B. cereus group members. This model resulted in 100% correct identification for the training set and 99.5% correct identification overall. The established ANN was applied to investigate the composition of B. cereus group members in soil, as a natural habitat of B. cereus, and in food samples originating from foodborne outbreaks. These analyses revealed a high complexity of B. cereus group populations, not only in soil samples but also in the samples from the foodborne outbreaks, highlighting the importance of taking multiple isolates from samples implicated in food poisonings. Notable, in contrast to the soil samples, no bacteria belonging to the psychrotolerant B. cereus group members were detected in the food samples linked to foodborne outbreaks, while the overall abundancy of B. thuringiensis did not significantly differ between the sample categories. None of the isolates was classified as B. cytotoxicus, fostering the hypothesis that the latter species is linked to very specific ecological niches. Overall, our work shows that machine learning assisted (FTIR) spectroscopy is suitable for identification of B. cereus group members in routine diagnostics and outbreak investigations. In addition, it is a promising tool to explore the natural habitats of B. cereus group, such as soil

From genome to toxicity: a combinatory approach highlights the complexity of enterotoxin production in Bacillus cereus

In recent years Bacillus cereus has gained increasing importance as a food poisoning pathogen. It is the eponymous member of the Bacillus cereus sensu lato group that consists of eight closely related species showing impressive diversity of their pathogenicity. The high variability of cytotoxicity and the complex regulatory network of enterotoxin expression have complicated efforts to predict the toxic potential of new Bacillus cereus isolates. In this study, comprehensive analyses of enterotoxin gene sequences, transcription, toxin secretion and cytotoxicity were performed. For the first time, these parameters were compared in a whole set of Bacillus cereus strains representing isolates of different origin (food or food poisoning outbreaks) and of different toxic potential (enteropathogenic and apathogenic) to elucidate potential starting points of strain-specific differential toxicity. While toxin gene sequences were highly conserved and did not allow for differentiation between high and low toxicity strains, comparison of nheB and hblD enterotoxin gene transcription and Nhe and Hbl protein titers revealed not only strain-specific differences but also incongruence between toxin gene transcripts and toxin protein levels. With one exception all strains showed comparable capability of protein secretion and so far, no secretion patterns specific for high and low toxicity strains were identified. These results indicate that enterotoxin expression is more complex than expected, possibly involving the orchestrated interplay of different transcriptional regulator proteins, as well as posttranscriptional and posttranslational regulatory mechanisms plus additional influences of environmental conditions