A cellular logic circuit for the detection of bacterial pore-forming toxins

We present a cellular logic circuit for deciphering the profiles of toxin production in B. cereus, using multiple readout techniques based on the pore formation on the cell membrane. This new assay enables the simultaneous detection of seven biomarkers in pathogenic strains from various samples

Complex Formation between NheB and NheC Is Necessary to Induce Cytotoxic Activity by the Three-Component Bacillus cereus Nhe Enterotoxin.

The nonhemolytic enterotoxin (Nhe) is known as a major pathogenicity factor for the diarrheal type of food poisoning caused by Bacillus cereus. The Nhe complex consists of NheA, NheB and NheC, all of them required to reach maximum cytotoxicity following a specific binding order on cell membranes. Here we show that complexes, formed between NheB and NheC under natural conditions before targeting the host cells, are essential for toxicity in Vero cells. To enable detection of NheC and its interaction with NheB, monoclonal antibodies against NheC were established and characterized. The antibodies allowed detection of recombinant NheC in a sandwich immunoassay at levels below 10 ng ml(-1), but no or only minor amounts of NheC were detectable in natural culture supernatants of B. cereus strains. When NheB- and NheC-specific monoclonal antibodies were combined in a sandwich immunoassay, complexes between NheB and NheC could be demonstrated. The level of these complexes was directly correlated with the relative concentrations of NheB and NheC. Toxicity, however, showed a bell-shaped dose-response curve with a plateau at ratios of NheB and NheC between 50:1 and 5:1. Both lower and higher ratios between NheB and NheC strongly reduced cytotoxicity. When the ratio approached an equimolar ratio, complex formation reached its maximum resulting in decreased binding of NheB to Vero cells. These data indicate that a defined level of NheB-NheC complexes as well as a sufficient amount of free NheB is necessary for efficient cell binding and toxicity. Altogether, the results of this study provide evidence that the interaction of NheB and NheC is a balanced process, necessary to induce, but also able to limit the toxic action of Nhe

Recent Developments in Antibody-Based Assays for the Detection of Bacterial Toxins

Considering the urgent demand for rapid and accurate determination of bacterial toxins and the recent promising developments in nanotechnology and microfluidics, this review summarizes new achievements of the past five years. Firstly, bacterial toxins will be categorized according to their antibody binding properties into low and high molecular weight compounds. Secondly, the types of antibodies and new techniques for producing antibodies are discussed, including poly- and mono-clonal antibodies, single-chain variable fragments (scFv), as well as heavy-chain and recombinant antibodies. Thirdly, the use of different nanomaterials, such as gold nanoparticles (AuNPs), magnetic nanoparticles (MNPs), quantum dots (QDs) and carbon nanomaterials (graphene and carbon nanotube), for labeling antibodies and toxins or for readout techniques will be summarized. Fourthly, microscale analysis or minimized devices, for example microfluidics or lab-on-a-chip (LOC), which have attracted increasing attention in combination with immunoassays for the robust detection or point-of-care testing (POCT), will be reviewed. Finally, some new materials and analytical strategies, which might be promising for analyzing toxins in the near future, will be shortly introduced

Versatile antibody-sensing Boolean logic for the simultaneous detection of multiple bacterial toxins

We present an OR gate based on monoclonal antibodies for the simultaneous detection of multiple toxins in a single tube. To further simplify the operating procedure, the Boolean rule of simplification was used to guide the selection of a marker toxin among the natural toxin profiles

The Mutation Glu151Asp in the B-Component of the Bacillus cereus Non-Hemolytic Enterotoxin (Nhe) Leads to a Diverging Reactivity in Antibody-Based Detection Systems

The ability of Bacillus cereus to cause foodborne toxicoinfections leads to increasing concerns regarding consumer protection. For the diarrhea-associated enterotoxins, the assessment of the non-hemolytic enterotoxin B (NheB) titer determined by a sandwich enzyme immunoassay (EIA) correlates best with in vitro cytotoxicity. In general, the regulation of enterotoxin expression of B. cereus is a coordinately-regulated process influenced by environmental, and probably also by host factors. As long as these factors are not completely understood, the currently-applied diagnostic procedures are based on indirect approaches to assess the potential virulence of an isolate. To date, sandwich EIA results serve as a surrogate marker to categorize isolates as either potentially low or highly toxic. Here, we report on a single amino acid exchange in the NheB sequence leading to an underestimation of the cytotoxic potential in a limited number of strains. During the screening of a large panel of B. cereus isolates, six showed uncommon features with low sandwich EIA titers despite high cytotoxicity. Sequence analysis revealed the point-mutation (Glu)151(Asp) in the potential binding region of the capture antibody. Application of this antibody also results in low titers in an indirect EIA format and shows variable detection intensities in Western-immunoblots. A commercially-available assay based on a lateral flow device detects all strains correctly as NheB producers in a qualitative manner. In conclusion, isolates showing low NheB titers should additionally be assayed in an indirect EIA or for their in vitro cytotoxicity to ensure a correct classification as either low or highly toxic

Consumed Foodstuffs Have a Crucial Impact on the Toxic Activity of Enteropathogenic Bacillus cereus

Enteropathogenic Bacillus cereus cause diarrhea due to the production of enterotoxins in the intestine. To start this process, spores have to be ingested together with contaminated food and survive the stomach passage. In this study, the influence of consumed foodstuffs on spore survival as well as on cytotoxicity toward colon epithelial cells was investigated. Spore survival of 20 enteropathogenic and apathogenic B. cereus strains during simulated stomach passage was highly strain-specific and did not correlate with the toxic potential. Survival of three tested strains was strain-specifically altered by milk products. Whereas milk, a follow-on formula and rice pudding had only little influence, spores seemed to be protected by milk products with high fat content such as whipped cream and mascarpone. Furthermore, tested milk products decreased the toxic activity of three B. cereus strains toward CaCo-2 cells. Investigating the individual components, lactoferrin, a skim milk powder and vitamins C, B5 and A showed the most inhibiting effects. On the other hand, biotin, vitamin B3 and another skim milk powder even enhanced cytotoxicity. Further studies suggested that these inhibiting effects result only partially from inhibiting cell binding, but rather from blocking the interaction between the single enterotoxin components

Effect of High Pressure and Heat on Bacterial Toxins

Even though the inactivation of microorganisms by high pressure treatment is a subject of intense investigations, the effect of high pressure on bacterial toxins has not been studied so far. In this study, the influence of combined pressure/temperature treatment (0.1 to 800 MPa and 5 to 121 °C) on bacterial enterotoxins was determined. Therefore, heat-stable enterotoxin (STa) of cholera toxin (CT) from Vibrio cholerae, staphylococcal enterotoxins A-E, haemolysin BL (HBL) from Bacillus cereus, and Escherichia coli (STa) were subjected to different treatment schemes. Structural alterations were monitored in enzyme immunoassays (EIAs). Cytotoxicity of the pressure treated supernatant of toxigenic B. cereus DSM 4384 was investigated with Vero cells. High pressure of 200 to 800 MPa at 5 °C leads to a slight increase of the reactivity of the STa of E. coli. However, reactivity decreased at 800 MPa and 80 °C to (66±21) % after 30 min and to (44±0.3) % after 128 min. At ambient pressure no decrease in EIA reactivity could be observed after 128 min. Pressurization (0.1 to 800 MPa) of heat stable monomeric staphylococcal toxins at 5 and 20 °C showed no effect. A combined heat (80 °C) and pressure (0.1 to 800 MPa) treatment lead to a decrease in the immuno-reactivity to 20 % of its maximum. For cholera toxin a significant loss in latex agglutination was observable only at 80 °C and 800 MPa for holding times higher than 20 min. Interestingly, the immuno-reactivity of B. cereus HBL toxin increased with the increase of pressure (182 % at 800 MPa, 30 °C), and high pressure showed only minor effects on cytotoxicity to Vero cells. Our results indicate that pressurization can increase inactivation observed by heat treatment, and combined treatments may be effective at lower temperatures and/or shorter incubation time

Effect of High Pressure and Heat on Bacterial Toxins

Even though the inactivation of microorganisms by high pressure treatment is a subject of intense investigations, the effect of high pressure on bacterial toxins has not been studied so far. In this study, the influence of combined pressure/temperature treatment (0.1 to 800 MPa and 5 to 121 °C) on bacterial enterotoxins was determined. Therefore, heat-stable enterotoxin (STa) of cholera toxin (CT) from Vibrio cholerae, staphylococcal enterotoxins A-E, haemolysin BL (HBL) from Bacillus cereus, and Escherichia coli (STa) were subjected to different treatment schemes. Structural alterations were monitored in enzyme immunoassays (EIAs). Cytotoxicity of the pressure treated supernatant of toxigenic B. cereus DSM 4384 was investigated with Vero cells. High pressure of 200 to 800 MPa at 5 °C leads to a slight increase of the reactivity of the STa of E. coli. However, reactivity decreased at 800 MPa and 80 °C to (66±21) % after 30 min and to (44±0.3) % after 128 min. At ambient pressure no decrease in EIA reactivity could be observed after 128 min. Pressurization (0.1 to 800 MPa) of heat stable monomeric staphylococcal toxins at 5 and 20 °C showed no effect. A combined heat (80 °C) and pressure (0.1 to 800 MPa) treatment lead to a decrease in the immuno-reactivity to 20 % of its maximum. For cholera toxin a significant loss in latex agglutination was observable only at 80 °C and 800 MPa for holding times higher than 20 min. Interestingly, the immuno-reactivity of B. cereus HBL toxin increased with the increase of pressure (182 % at 800 MPa, 30 °C), and high pressure showed only minor effects on cytotoxicity to Vero cells. Our results indicate that pressurization can increase inactivation observed by heat treatment, and combined treatments may be effective at lower temperatures and/or shorter incubation time

Recent Developments in Antibody-Based Assays for the Detection of Bacterial Toxins

Considering the urgent demand for rapid and accurate determination of bacterial toxins and the recent promising developments in nanotechnology and microfluidics, this review summarizes new achievements of the past five years. Firstly, bacterial toxins will be categorized according to their antibody binding properties into low and high molecular weight compounds. Secondly, the types of antibodies and new techniques for producing antibodies are discussed, including poly- and mono-clonal antibodies, single-chain variable fragments (scFv), as well as heavy-chain and recombinant antibodies. Thirdly, the use of different nanomaterials, such as gold nanoparticles (AuNPs), magnetic nanoparticles (MNPs), quantum dots (QDs) and carbon nanomaterials (graphene and carbon nanotube), for labeling antibodies and toxins or for readout techniques will be summarized. Fourthly, microscale analysis or minimized devices, for example microfluidics or lab-on-a-chip (LOC), which have attracted increasing attention in combination with immunoassays for the robust detection or point-of-care testing (POCT), will be reviewed. Finally, some new materials and analytical strategies, which might be promising for analyzing toxins in the near future, will be shortly introduced

Multiplexed Lateral Flow Test for Detection and Differentiation of Cronobacter sakazakil Serotypes 01 and 02

The ubiquitous and opportunistic pathogen Cronobacter sakazakii is responsible for severe meningitis, sepsis, and necrotizing enterocolitis in neonates and infants associated with ingestion of contaminated powdered infant formula (PIF). The current ISO method for isolation and detection of Cronobacter spp. is laborious, time-consuming and expensive. In this study, a multiplexed lateral flow test strip was developed to rapidly detect and simultaneously serotype O1 and O2 C. sakazakil serotypes. The assay is based on two monoclonal antibodies (MAb) that specifically bind to the lipopolysaccharides (LPS) of these pathogens. The test strip provides results very quickly;C. sakazakii could be detected in pure culture within 15 min with a sensitivity of 107 CFU/ml. After non-selective enrichment for 18 h as low as one Cronobacter cell per g PIF could be detected. Moreover, the established lateral flow assay (LFA) offers excellent specificity showing no cross-reactivity with other C. sakazakii serotypes, Cronobacter species or Enterobacteriaceae tested. These characteristics, together with several advantages such as speed, simplicity in performance, low analysis cost, and no requirement of specialized skills or sophisticated equipment make the developed multiplexed LFA suitable for reliable detection and serotyping of C. sakazakii serotypes O1 and O2