Antimicrobial Ionic Liquids: Ante-Mortem Mechanisms of Pathogenic EPEC and MRSA Examined by FTIR Spectroscopy.

Ionic liquids (ILs) have gained considerable attention due to their versatile and designable properties. ILs show great potential as antibacterial agents, but understanding the mechanism of attack on bacterial cells is essential to ensure the optimal design of IL-based biocides. The final aim is to achieve maximum efficacy while minimising toxicity and preventing resistance development in target organisms. In this study, we examined a dose-response analysis of ILs' antimicrobial activity against two pathogenic bacteria with different Gram types in terms of molecular responses on a cellular level using Fourier-transform infrared (FTIR) spectroscopy. In total, 18 ILs with different antimicrobial active motifs were evaluated on the Gram-negative enteropathogenic Escherichia coli (EPEC) and Gram-positive methicillin-resistant Staphylococcus aureus (MRSA). The results showed that most ILs impact bacterial proteins with increasing concentration but have a minimal effect on cellular membranes. Dose-response spectral analysis revealed a distinct ante-mortem response against certain ILs for MRSA but not for EPEC. We found that at sub-lethal concentrations, MRSA actively changed their membrane composition to counteract the damaging effect induced by the ILs. This suggests a new adaptive mechanism of Gram-positive bacteria against ILs and demonstrates the need for a better understanding before using such substances as novel antimicrobials.

A poisonous cocktail: interplay of cereulide toxin and its structural isomers in emetic Bacillus cereus.

Food intoxications evoked by emetic Bacillus cereus strains constitute a serious threat to public health, leading to emesis and severe organ failure. The emetic peptide toxin cereulide, assembled by the non-ribosomal peptide synthetase CesNRPS, cannot be eradicated from contaminated food by usual hygienic measures due to its molecular size and structural stability. Next to cereulide, diverse chemical variants have been described recently that are produced concurrently with cereulide by CesNRPS. However, the contribution of these isocereulides to the actual toxicity of emetic B. cereus, which produces a cocktail of these toxins in a certain ratio, is still elusive. Since cereulide isoforms have already been detected in food remnants from foodborne outbreaks, we aimed to gain insights into the composition of isocereulides and their impact on the overall toxicity of emetic B. cereus. The amounts and ratios of cereulide and isocereulides were determined in B. cereus grown under standard laboratory conditions and in a contaminated sample of fried rice balls responsible for one of the most severe food outbreaks caused by emetic B. cereus in recent years. The ratios of variants were determined as robust, produced either under laboratory or natural, food-poisoning conditions. Examination of their actual toxicity in human epithelial HEp2-cells revealed that isocereulides A-N, although accounting for only 10% of the total cereulide toxins, were responsible for about 40% of the total cytotoxicity. An this despite the fact that some of the isocereulides were less cytotoxic than cereulide when tested individually for cytotoxicity. To estimate the additive, synergistic or antagonistic effects of the single variants, each cereulide variant was mixed with cereulide in a 1:9 and 1:1 binary blend, respectively, and tested on human cells. The results showed additive and synergistic impacts of single variants, highlighting the importance of including not only cereulide but also the isocereulides in routine food and clinical diagnostics to achieve a realistic toxicity evaluation of emetic B. cereus in contaminated food as well as in patient samples linked to foodborne outbreaks. Since the individual isoforms confer different cell toxicity both alone and in association with cereulide, further investigations are needed to fully understand their cocktail effect.

Prospective Study on the Influence of Occupational Hand Protection Products on the Efficacy of Hand Disinfection.

BACKGROUND: To prevent occupational skin diseases, employees are instructed to periodically apply hand protection products as a barrier to protect their hands from water, cleaning agents or other irritants. The aim of this work was to investigate whether bacteria present on the skin at the time of protection product application are enclosed underneath this protective layer, if they can be transferred to other surfaces and if a standard isopropanol-based skin disinfectant can nonetheless reduce the bacterial burden. METHODS: This prospective study was conducted in human volunteers based on the European Standard (EN 1500) to assess the burden of microorganisms before and after the application of various protection product formulations and subsequent hand disinfection. RESULTS: All protection products, with the exception of alcohol-based gels, enclosed bacteria underneath a lipid layer which could be transferred onto other surfaces. Still, the hand disinfectant efficiently reduced the bacteria burden. DISCUSSION: In occupations where proper hand hygiene is vital, alcohol-based gels might be the best option for the protection of the skin barrier as well as for reducing the contamination risk. CONCLUSION: An alcohol-based disinfection agent can dissolve the lipid film of protection products following the standard protocol for hygienic hand disinfection.

Diversity of Staphylococcus aureus associated with mastitis from dairy cows in Rwanda.

OBJECTIVES: The objective of the present study was to examine the diversity of Staphylococcus aureus from mastitis milk samples of cows in Rwanda. METHODS: A total of 1080 quarter milk samples from 279 dairy cows were collected in 80 different farms from all five provinces of Rwanda. In total, 135 S. aureus isolates were obtained and subjected to genotyping (spa typing, DNA microarray, whole-genome sequencing (WGS)), antimicrobial susceptibility testing (AST) and phenotypic profiling by Fourier Transform Infrared (FTIR) spectroscopy (including capsular serotyping). RESULTS: Resistance to penicillin and/or tetracycline was most frequently observed. Ten sequence types (STs) (ST1, ST151, ST152, ST5477, ST700, ST7110, ST7983, ST7984, ST8320, ST97) belonging to seven clonal complexes (CCs) (CC1, CC130, CC152, CC3591, CC3666, CC705, CC97) were detected. The Panton-Valentine leukocidin (PVL) genes (lukF-PV/lukS-PV), the bovine leukocidin genes (lukM/lukF-P83) and the human and bovine toxic shock syndrome toxin gene tst-1 variants were detected. FTIR-based capsular serotyping showed CC-specific differences. Most CC97 (cap5 allele) isolates were primarily nonencapsulated (82%), whereas isolates of CC3591 and CC3666 (cap8 allele) were mostly encapsulated (86.4% and 57.8%, respectively). Our results underline the widespread global distribution of cattle-adapted CC97. CONCLUSION: The presence of CC3591 and CC3666 in bovine mastitis suggests an important role in cattle health and dairy production in Rwanda. The results of the present study support the need for a rigorous One-Health Surveillance program of the bovine-human interface.

Diversity of Staphylococcus aureus isolated from nares of ruminants.

AIMS: To examine the diversity of Staphylococcus aureus isolated from nasal swabs of ruminants in Rwanda. METHODS AND RESULTS: A total of 454 nasal swabs from 203 cows, 170 goats, and 81 sheep were examined for the presence of S. aureus, and 30 S. aureus isolates were detected and characterized pheno- and genotypically. Resistance to penicillin and/or tetracycline was observed. The isolates were assigned to eight different spa types (t21057 (novel), t10103, t18853, t20842, t318, t355, t458, and t9432) belonging to six clonal complexes (CCs) (CC152, CC30, CC3591, CC3666, CC522, and CC97). Panton-Valentine leukocidin (PVL) genes (lukF-PV/lukS-PV), the bovine leukocidin genes (lukM/lukF-P83), and the human and bovine variants of the toxic shock syndrome toxin gene tst-1 variants were detected. CONCLUSION: These findings demonstrate that the nares of ruminants in Rwanda are colonized with mastitis-associated S. aureus, including lineages that are also carried by humans, underscoring the zoonotic risk, especially for livestock keepers. These results highlight the crucial importance of hygiene measures when handling livestock.

The toxicological spectrum of the Bacillus cereus toxin cereulide points towards niche-specific specialisation.

Most microbes share their environmental niches with very different forms of life thereby engaging in specialised relationships to enable their persistence. The bacterium Bacillus cereus occurs ubiquitously in the environment with certain strain backgrounds causing foodborne and opportunistic infections in humans. The emetic lineage of B. cereus is capable of producing the toxin cereulide, which evokes emetic illnesses. Although food products favouring the accumulation of cereulide are known, the ecological role of cereulide and the environmental niche of emetic B. cereus remain elusive. To better understand the ecology of cereulide-producing B. cereus, we systematically assayed the toxicological spectrum of cereulide on a variety of organisms belonging to different kingdoms. As cereulide is a potassium ionophore, we further tested the effect of environmental potassium levels on the action of cereulide. We found that adverse effects of cereulide exposure are species-specific, which can be exacerbated with increased environmental potassium. Additionally, we demonstrate that cereulide is produced within an insect cadaver indicating its potential ecological function for a saprophytic lifestyle. Collectively, distinct cereulide susceptibilities of other organisms may reflect its role in enabling competitive niche specialization of emetic B. cereus.

Bacillus cereus extracellular vesicles act as shuttles for biologically active multicomponent enterotoxins.

BACKGROUND: Extracellular vesicles (EVs) from Gram-positive bacteria have gained considerable importance as a novel transport system of virulence factors in host-pathogen interactions. Bacillus cereus is a Gram-positive human pathogen, causing gastrointestinal toxemia as well as local and systemic infections. The pathogenicity of enteropathogenic B. cereus has been linked to a collection of virulence factors and exotoxins. Nevertheless, the exact mechanism of virulence factor secretion and delivery to target cells is poorly understood. RESULTS: Here, we investigate the production and characterization of enterotoxin-associated EVs from the enteropathogenic B. cereus strain NVH0075-95 by using a proteomics approach and studied their interaction with human host cells in vitro. For the first time, comprehensive analyses of B. cereus EV proteins revealed virulence-associated factors, such as sphingomyelinase, phospholipase C, and the three-component enterotoxin Nhe. The detection of Nhe subunits was confirmed by immunoblotting, showing that the low abundant subunit NheC was exclusively detected in EVs as compared to vesicle-free supernatant. Cholesterol-dependent fusion and predominantly dynamin-mediated endocytosis of B. cereus EVs with the plasma membrane of intestinal epithelial Caco2 cells represent entry routes for delivery of Nhe components to host cells, which was assessed by confocal microscopy and finally led to delayed cytotoxicity. Furthermore, we could show that B. cereus EVs elicit an inflammatory response in human monocytes and contribute to erythrocyte lysis via a cooperative interaction of enterotoxin Nhe and sphingomyelinase. CONCLUSION: Our results provide insights into the interaction of EVs from B. cereus with human host cells and add a new layer of complexity to our understanding of multicomponent enterotoxin assembly, offering new opportunities to decipher molecular processes involved in disease development. Video Abstract.

Plasmid - Chromosome interplay in natural and non-natural hosts: global transcription study of three Bacillus cereus group strains carrying pCER270 plasmid.

The Bacillus cereus group comprises genetically related Gram-positive spore-forming bacteria that colonize a wide range of ecological niches and hosts. Despite their high degree of genome conservation, extrachromosomal genetic material diverges between these species. The discriminating properties of the B. cereus group strains are mainly due to plasmid-borne toxins, reflecting the importance of horizontal gene transfers in bacterial evolution and species definition. To investigate how a newly acquired megaplasmid can impact the transcriptome of its host, we transferred the pCER270 from the emetic B. cereus strains to phylogenetically distant B. cereus group strains. RNA-sequencing experiments allowed us to determine the transcriptional influence of the plasmid on host gene expression and the impact of the host genomic background on the pCER270 gene expression. Our results show a transcriptional cross-regulation between the megaplasmid and the host genome. pCER270 impacted carbohydrate metabolism and sporulation genes expression, with a higher effect in the natural host of the plasmid, suggesting a role of the plasmid in the adaptation of the carrying strain to its environment. In addition, the host genomes also modulated the expression of pCER270 genes. Altogether, these results provide an example of the involvement of megaplasmids in the emergence of new pathogenic strains.

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.

Prevalence of the SigB-Deficient Phenotype among Clinical Staphylococcus aureus Isolates Linked to Bovine Mastitis.

Phenotypic adaptation has been associated with persistent, therapy-resistant Staphylococcus aureus infections. Recently, we described within-host evolution towards a Sigma factor B (SigB)-deficient phenotype in a non-human host, a naturally infected dairy cow with chronic, persistent mastitis. However, to our knowledge, the prevalence of SigB deficiency among clinical S. aureus isolates remains unknown. In this study, we screened a collection of bovine mastitis isolates for phenotypic traits typical for SigB deficiency: decreased carotenoid pigmentation, increased proteolysis, secretion of α-hemolysin and exoproteins. Overall, 8 out of 77 (10.4%) isolates of our bovine mastitis collection exhibited the SigB-deficient phenotype. These isolates were assigned to various clonal complexes (CC8, CC9, CC97, CC151, CC3666). We further demonstrated a strong positive correlation between asp23-expression (a marker of SigB activity) and carotenoid pigmentation (r = 0.6359, p = 0.0008), underlining the role of pigmentation as a valuable predictor of the functional status of SigB. Sequencing of the sigB operon (mazEF-rsbUVW-sigB) indicated the phosphatase domain of the RsbU protein as a primary target of mutations leading to SigB deficiency. Indeed, by exchanging single nucleotides in rsbU, we could either induce SigB deficiency or restore the SigB phenotype, demonstrating the pivotal role of RsbU for SigB functionality. The data presented highlight the clinical relevance of SigB deficiency, and future studies are needed to exploit its role in staphylococcal infections.

From Gut to Blood: Spatial and Temporal Pathobiome Dynamics during Acute Abdominal Murine Sepsis.

Abdominal sepsis triggers the transition of microorganisms from the gut to the peritoneum and bloodstream. Unfortunately, there is a limitation of methods and biomarkers to reliably study the emergence of pathobiomes and to monitor their respective dynamics. Three-month-old CD-1 female mice underwent cecal ligation and puncture (CLP) to induce abdominal sepsis. Serial and terminal endpoint specimens were collected for fecal, peritoneal lavage, and blood samples within 72 h. Microbial species compositions were determined by NGS of (cell-free) DNA and confirmed by microbiological cultivation. As a result, CLP induced rapid and early changes of gut microbial communities, with a transition of pathogenic species into the peritoneum and blood detected at 24 h post-CLP. NGS was able to identify pathogenic species in a time course-dependent manner in individual mice using cfDNA from as few as 30 microliters of blood. Absolute levels of cfDNA from pathogens changed rapidly during acute sepsis, demonstrating its short half-life. Pathogenic species and genera in CLP mice significantly overlapped with pathobiomes from septic patients. The study demonstrated that pathobiomes serve as reservoirs following CLP for the transition of pathogens into the bloodstream. Due to its short half-life, cfDNA can serve as a precise biomarker for pathogen identification in blood.

Impact of a Novel PagR-like Transcriptional Regulator on Cereulide Toxin Synthesis in Emetic Bacillus cereus.

The emetic type of foodborne disease caused by Bacillus cereus is produced by the small peptide toxin cereulide. The genetic locus encoding the Ces nonribosomal peptide synthetase (CesNRPS) multienzyme machinery is located on a 270 kb megaplasmid, designated pCER270, which shares its backbone with the Bacillus anthracis toxin plasmid pXO1. Although the ces genes are plasmid-borne, the chromosomally encoded pleiotropic transcriptional factors CodY and AbrB are key players in the control of ces transcription. Since these proteins only repress cereulide synthesis during earlier growth phases, other factors must be involved in the strict control of ces expression and its embedment in the bacterial life cycle. In silico genome analysis revealed that pCER270 carries a putative ArsR/SmtB family transcription factor showing high homology to PagR from B. anthracis. As PagR plays a crucial role in the regulation of the protective antigen gene pagA, which forms part of anthrax toxin, we used a gene-inactivation approach, combined with electrophoretic mobility shift assays and a bacterial two-hybrid system for dissecting the role of the PagR homologue PagRBc in the regulation of cereulide synthesis. Our results highlight that the plasmid-encoded transcriptional regulator PagRBc plays an important role in the complex and multilayered process of cereulide synthesis.

Proteomic and immunoproteomic insights into the exoproteome of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia.

Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae affects pig health status and the swine industry worldwide. Despite the extensive number of studies focused on A. pleuropneumoniae infection and vaccine development, a thorough analysis of the A. pleuropneumoniae exoproteome is still missing. Using a complementary approach of quantitative proteomics and immunoproteomics we gained an in-depth insight into the A. pleuropneumoniae serotype 2 exoproteome, which provides the basis for future functional studies. Label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed 593 exoproteins, of which 104 were predicted to be virulence factors. The RTX toxins ApxIIA and ApxIIIA -were found to be the most abundant proteins in the A. pleuropneumoniae serotype 2 exoproteome. Furthermore, the ApxIVA toxin was one of the proteins showing the highest abundance, although ApxIVA is commonly assumed to be expressed exclusively in vivo. Our study revealed several antigens, including proteins with moonlight functions, such as the elongation factor (EF)-Tu, and proteins linked to specific metabolic traits, such as the maltodextrin-binding protein MalE, that warrant future functional characterization and might present potential targets for novel therapeutics and vaccines. Our Ig-classes specific serological proteome analysis (SERPA) approach allowed us to explore the development of the host humoral immune response over the course of the infection. These SERPAs pinpointed proteins that might play a key role in virulence and persistence and showed that the immune response to the different Apx toxins is distinct. For instance, our results indicate that the ApxIIIA toxin has properties of a thymus-independent antigen, which should be studied in more detail.

Cereulide and Deoxynivalenol Increase LC3 Protein Levels in HepG2 Liver Cells.

Food contaminants of bacterial or fungal origin frequently contaminate staple foods to various extents. Among others, the bacterial toxin cereulide (CER) and the mycotoxin deoxynivalenol (DON) co-occur in a mixed diet and are absorbed by the human body. Both toxins exert dis-tinctive mitotoxic potential. As damaged mitochondria are removed via autophagy, mitochondrial and lysosomal toxicity were assessed by applying low doses of single and combined toxins (CER 0.1-50 ng/mL; DON 0.01-5 µg/mL) to HepG2 liver cells. In addition to cytotoxicity assays, RT-qPCR was performed to investigate genes involved in lysosomal biogenesis and autophagy. CER and DON caused significant cytotoxicity on HepG2 cells after 5 and 24 h over a broad concentration range. CER, alone and in combination with DON, increased the transcription of the autophagy related genes coding for the microtubule associated protein 1A/1B light chain 3 (LC3) and sequestome 1 (SQSTM1) as well as LC3 protein expression which was determined using immunocytochemistry. DON increased LC3 protein expression without induction of gene transcription, hence it seems plausible that CER and DON act on different pathways. The results support the hypothesis that CER induces autophagy via the LC3 pathway and damaged mitochondria are therefore eliminated.

Bacillus cereus Toxin Repertoire: Diversity of (Iso)cereulide(s).

The emetic Bacillus cereus toxin cereulide (1) poses a significant safety risk in the food industry, causing emesis and nausea after consumption of contaminated foods. Analogously to cereulide, the structures of various isocereulides, namely, isocereulides A-G, have been recently reported and could also be identified in B. cereus-contaminated food samples. The HPLC fractionation of B. cereus extracts allows us to isolate additional isocereulides. By applying MSn sequencing, post-hydrolytic dipeptide, amino acid and α-hydroxy acid analyses using UPLC-ESI-TOF-MS to purify the analytes, seven new isocereulides H-N (2-8) could be elucidated in their chemical structures. The structure elucidation was supported by one-dimensional and two-dimensional NMR spectra of the isocereulides H (2), K (5), L and N (6 + 8) and M (7). The toxicity of 2-8 was investigated in a HEp-2 cell assay to determine their respective 50% effective concentration (EC50). Thus, 2-8 exhibited EC50 values ranging from a 0.4- to 1.4-fold value compared to cereulide (1). Missing structure-activity correlations indicate the necessity to determine the toxic potential of all naturally present isocereulides as single compounds to be able to perform a thorough toxicity evaluation of B. cereus-contaminated foods in the future.

Performance Testing of Bacillus cereus Chromogenic Agar Media for Improved Detection in Milk and Other Food Samples.

In this study, the performance of four alternative selective chromogenic B. cereus agar was compared to the reference mannitol-yolk polymyxin (MYP) agar (ISO 7932) using inclusion and exclusion test strains (n = 110) and by analyzing naturally contaminated milk and other food samples (n = 64). Subsequently, the panC group affiliation and toxin gene profile of Bacillus cereus senso lato (s.l.) isolates were determined. Our results corroborate that the overall best performing media CHROMagar™ B. cereus (93.6% inclusivity; 82.7% exclusivity) and BACARA® (98.2% inclusivity, 62.7% exclusivity) are more sensitive and specific compared to Brilliance™ B. cereus, MYP and ChromoSelect Bacillus Agar. Both media allow unequivocal detection of B. cereus with low risks of misidentification. Media containing ß-D-glucosidase for the detection of presumptive B. cereus may form atypical colony morphologies resulting in a false negative evaluation of the sample. Naturally contaminated samples presented high numbers of background flora, while numbers of presumptive B. cereus were below the detection limit (<10 CFU g-1 or mL-1). Recovery after freezing resulted in the highest detection of B. cereus s.l. on BACARA® (57.8%), CHROMagar™ B. cereus (56.3%) and MYP agar (54.7%). The panC/toxin profile combination IV/A was the most abundant (33.0%), followed by III/F (21.7%) and VI/C (10.4%). More panC and toxin combinations were present in 15.6% of samples when reanalyzed after freezing. In order to improve detection and confirmation of B. cereus s.l. in food samples, we recommend the parallel use of two complementary selective media followed by molecular characterization (e.g., panC typing combined with toxin gene profiling). When determining psychrotolerant or thermophilic members of the B. cereus group, the selective agar media should additionally be incubated at appropriate temperatures (5 °C, ≥45 °C). If high-risk toxin genes (e.g., ces or cytK-1) are detected, the strain-specific ability to produce toxin should be examined to decisively assess risk.