Microfluidic chip analysis of outer membrane proteins responsible for serological cross-reaction between three Gram-negative bacteria: Proteus morganii O34, Escherichia coli O111 and Salmonella Adelaide O35 (original) (raw)
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Bangladesh Journal of Veterinary Medicine, 2009
The study was conducted to compare the protein patterns among some Salmonella serovars and E. coli using Two Dimensional Polyacrylamide Gel Electophoresis. The Two Dimensional Polyacrylamide Gel Electophoresis showed a 37.81 kDa well separated protein spots with all Salmonella serovars at the same time with E. coli a 36.5 kDa protein. However, these protein spots of Two Dimensional Polyacrylamide Gel Electophoresis were further tested with Immunoblotting analysis with specific antiserum against Salmonella typhimurium infected chicks. All selected Salmonella serovars successfully identified a common 37.81 kDa protein whereas E. coli spots identified as 36.5 kDa protein instead of 37.81 kDa. As a further monitoring of these proteins as to check the homogeneity and heterogeneity for N-terminal amino acid sequencing, the specific protein bands from all Salmonella serovars and E. coli were excised, purified and subjected to sequence analysis. The amino acid sequence alignment showed the 37.81 kDa proteins of some Salmonella serovars were identical or homologous among the Salmonella serovars. The N-terminal amino acid alignments of the 37.81 kDa proteins were determined as alanineglutamine-valine-isoleucine-asparagine-threonine-asparagine. On the other hand, the N-terminal amino acid alignment of the 36.5 kDa protein of E. cloi ACLD2201 was found to be heterologous as alanine-proline-lysine-aspartic acid-aspararginethreonine-tryptophan. The findings of this study can be concluded that the 37.81 kDa protein of some Salmonella serovars and 36.5 kDa protein of E. coli were completely different though there is some identity of these organisms due to the presence of Enterobacterial common antigen.
Lab on a Chip, 2012
The recent outbreaks of a lethal E. coli strain in Germany have aroused renewed interest in developing rapid, specific and accurate systems for detecting and characterizing bacterial pathogens in suspected contaminated food and/or water supplies. To address this need, we have designed, fabricated and tested an integrated modular-based microfluidic system and the accompanying assay for the strain-specific identification of bacterial pathogens. The system can carry out the entire molecular processing pipeline in a single disposable fluidic cartridge and detect single nucleotide variations in selected genes to allow for the identification of the bacterial species, even its strain with high specificity. The unique aspect of this fluidic cartridge is its modular format with task-specific modules interconnected to a fluidic motherboard to permit the selection of the target material. In addition, to minimize the amount of finishing steps for assembling the fluidic cartridge, many of the functional components were produced during the polymer molding step used to create the fluidic network. The operation of the cartridge was provided by electronic, mechanical, optical and hydraulic controls located off-chip and packaged into a small footprint instrument (1 ft 3). The fluidic cartridge was capable of performing cell enrichment, cell lysis, solid-phase extraction (SPE) of genomic DNA, continuous flow (CF) PCR, CF ligase detection reaction (LDR) and universal DNA array readout. The cartridge was comprised of modules situated on a fluidic motherboard; the motherboard was made from polycarbonate, PC, and used for cell lysis, SPE, CF PCR and CF LDR. The modules were taskspecific units and performed universal zip-code array readout or affinity enrichment of the target cells with both made from poly(methylmethacrylate), PMMA. Two genes, uidA and sipB/C, were used to discriminate between E. coli and Salmonella, and evaluated as a model system. Results † Electronic Supplementary Information (ESI) available. See
Surface display of Salmonella epitopes in Escherichia coli and Staphylococcus carnosus
Microbial Cell Factories, 2011
Background: Salmonella enterica serotype Enteritidis (SE) is considered to be one of the most potent pathogenic Salmonella serotypes causing food-borne disease in humans. Since a live bacterial vaccine based on surface display of antigens has many advantages over traditional vaccines, we have studied the surface display of the SE antigenic proteins, H:gm and SefA in Escherichia coli by the β-autotransporter system, AIDA. This procedure was compared to protein translocation in Staphylococcus carnosus, using a staphylococci hybrid vector earlier developed for surface display of other vaccine epitopes. Results: Both SefA and H:gm were translocated to the outer membrane in Escherichia coli. SefA was expressed to full length but H:gm was shorter than expected, probably due to a proteolytic cleavage of the N-terminal during passage either through the periplasm or over the membrane. FACS analysis confirmed that SefA was facing the extracellular environment, but this could not be conclusively established for H:gm since the N-terminal detection tag (His 6 ) was cleaved off. Polyclonal salmonella antibodies confirmed the sustained antibody-antigen binding towards both proteins. The surface expression data from Staphylococcus carnosus suggested that the H:gm and SefA proteins were transported to the cell wall since the detection marker was displayed by FACS analysis. Conclusion: Apart from the accumulated knowledge and the existence of a wealth of equipment and techniques, the results indicate the selection of E. coli for further studies for surface expression of salmonella antigens. Surface expression of the full length protein facing the cell environment was positively proven by standard analysis, and the FACS signal comparison to expression in Staphylococcus carnosus shows that the distribution of the surface protein on each cell was comparatively very narrow in E. coli, the E. coli outer membrane molecules can serve as an adjuvant for the surface antigenic proteins and multimeric forms of the SefA protein were detected which would probably be positive for the realisation of a strong antigenic property. The detection of specific and similar proteolytic cleavage patterns for both the proteins provides a further starting point for the investigation and development of the Escherichia coli AIDA autotransporter efficiency.
Infection and immunity, 1985
Antisera raised against several smooth and rough strains of Escherichia coli and Salmonella typhimurium were tested against lipopolysaccharides (LPS) of homologous and heterologous strains. The LPS were separated by sodium dodecyl sulfate-gel electrophoresis, transferred to nitrocellulose paper, and overlaid with antisera. The results showed that antisera raised against smooth strains reacted with high- as well as low-molecular-weight bands of their corresponding LPS and showed very few cross-reactions. Anti-E. coli J5 antiserum cross-reacted with few strains in the core region. But, anti-S. typhimurium Ra antiserum cross-reacted with many more strains. When these sera were absorbed with either the homologous- or a heterologous-positive strain, reactions were abolished. It appears that reactions of anti-E. coli J5 antiserum and anti-S. typhimurium Ra antiserum with homologous and heterologous strains were not due to the same antibody. This immunoblotting technique proved to be a use...
Journal of Immunological Methods, 2006
Bacterial cell surface antigens interact with the host immune system resulting in the production of antibodies. Detection of antibodies against surface antigens has applications in diagnosis of many bacterial infections, assessment of immune status and epidemiological studies. We developed a microarray platform, for antibody detection, by printing Gram-negative and Grampositive whole bacterial cells on nitrocellulose coated glass substrates. Antibody binding was detected using fluorophore labeled secondary antibodies. The sensitivity of antibody detection was found to be 0.1 Ag/ml. Using bacterial cell microarrays it was also possible to successfully detect antibodies against Francisella tularensis in canine serum samples declared positive for tularemia based on microagglutination antibody titer. Use of bacterial cells as the antigen source in immunoassays has the advantages of simulating in vivo presentation of surface antigens and also eliminating the need for antigen purification. The microarray format gives the added advantage of simultaneous detection of antibodies against multiple bacteria employing only small amounts of samples and reagents.
Molecular Determination of H Antigens of Salmonella by Use of a Microsphere-Based Liquid Array
Journal of Clinical Microbiology, 2011
Serotyping of Salmonella has been an invaluable subtyping method for epidemiologic studies for more than 70 years. The technical difficulties of serotyping, primarily in antiserum production and quality control, can be overcome with modern molecular methods. We developed a DNA-based assay targeting the genes encoding the flagellar antigens (fliC and fljB) of the Kauffmann-White serotyping scheme. Fifteen H antigens (H:z 35 , and -z 6 ), 5 complex major antigens (H:G, -EN, -Z4, -1, and -L) and 16 complex secondary antigens (H:2, -5, -6, -7, -f, -m/g,m, -m/m,t, -p, -s, -t/m,t, -v, -x, -z 15 , -z 24 , -z 28 , and -z 51 ) were targeted in the assay. DNA probes targeting these antigens were designed and evaluated on 500 isolates tested in parallel with traditional serotyping methods. The assay correctly identified 461 (92.2%) isolates based on the 36 antigens detected in the assay. Among the isolates considered correctly identified, 47 (9.4%) were partially serotyped because probes corresponding to some antigens in the strains were not in the assay, and 13 (2.6%) were monophasic or nonmotile strains that possessed flagellar antigen genes that were not expressed but were detected in the assay. The 39 (7.8%) strains that were not correctly identified possessed an antigen that should have been detected by the assay but was not. Apparent false-negative results may be attributed to allelic divergence. The molecular assay provided results that paralleled traditional methods with a much greater throughput, while maintaining the integrity of the Kauffmann-White serotyping scheme, thus providing backwards-compatible epidemiologic data. This assay should greatly enhance the ability of clinical and public health laboratories to serotype Salmonella.
Label-free biosensing of Salmonella enterica serovars at single-cell level
Journal of nanobiotechnology, 2017
The emerging nanotechnologies have greatly facilitated the development of label-free biosensors. The atomic force microscopy (AFM) has been used to study the molecular mechanism of the reactions for protein and aptamers. The surface plasmon resonance (SPR) have been used in fast detections of various pathogens such as bacteria. This study used both AFM and SPR to investigate the complex reactions between aptamers and outer membrane proteins (OMPs) on the surface of S. typhimurium. Two DNA aptamers were used for the label-free detections of S. typhimurium by AFM and SPR. The aptamers have specific binding affinities to the OMPs of S. typhimurium. At single-molecule level, the high resolution AFM topography and recognition images distinguished the OMPs on the bacteria surface, which is the first time the location of individual outer membrane protein have been determined on Salmonella surface. E. coli in the control experiments didn't generate recognition signals, which proved the ...
Analytical Chemistry, 2010
Low abundant (<100 cells mL-1) E. coli O157:H7 cells were isolated and enriched from environmental water samples using a microfluidic chip. The poly(methylmethacrylate), PMMA, chip contained 8 devices each equipped with 16 curvilinear high aspect ratio channels that were covalently decorated with polyclonal anti-O157 antibodies (pAb) and could search for rare cells through a pAb mediated process. The chip could process independently 8 different samples or one sample using 8 different parallel inputs to increase volume processing throughput. After cell enrichment, cells were released and enumerated using bench top real-time quantitative PCR, targeting genes which effectively discriminated the O157:H7 serotype from other non-pathogenic bacteria. The recovery of target cells from water samples was determined to be ~72%, and the limit-of-detection was found to be 6 colony forming units (cfu) using the slt1 gene as a reporter. We subsequently performed analysis of lake and waste water samples. The simplicity in manufacturing and ease of operation makes this device attractive for the selection of pathogenic species from a variety of water supplies suspected of containing bacterial pathogens at extremely low frequencies.
Application of Microfluidics for Bacterial Identification
Pharmaceuticals
Bacterial infections continue to pose serious public health challenges. Though anti-bacterial therapeutics are effective remedies for treating these infections, the emergence of antibiotic resistance has imposed new challenges to treatment. Often, there is a delay in prescribing antibiotics at initial symptom presentation as it can be challenging to clinically differentiate bacterial infections from other organisms (e.g., viruses) causing infection. Moreover, bacterial infections can arise from food, water, or other sources. These challenges have demonstrated the need for rapid identification of bacteria in liquids, food, clinical spaces, and other environments. Conventional methods of bacterial identification rely on culture-based approaches which require long processing times and higher pathogen concentration thresholds. In the past few years, microfluidic devices paired with various bacterial identification methods have garnered attention for addressing the limitations of convent...
2009 IEEE Sensors Applications Symposium, 2009
Salmonalla is one of the major causes of worldwide foodborne diseases. Conventional microbiological detection methods are time consuming and the recent signal transduction schemes based on fluorescence spectroscopy, surface plasmon resonance and others require specialized bulky and expensive instruments, which will increase the overall production cost in the food industry. This paper reports for the first time the use of microcellular trapping channels of oxidized macroporous silicon substrate with stable contacts directly from macroporous silicon using antibody-antigen binding method for label free, rapid, field deployable and inexpensive electrical detection of Salmonella Typhimurium by impedance measurements. Macroporous silicon is a regular array of pores of 1-2µm diameter which act as microcellular trapping medium for capture of bacteria. The preliminary reports show that a 2mm by 1mm electrode structure with a spacing of 1mm on oxidized macroporous silicon, without any optimization has been able to detect 10 3 CFU-10 7 CFU/ml of Salmonella Typhimurium using this microcellular trapping medium at a significantly lower processing cost and is comparable to much more sophisticated impedimetric measurements using interdigitated microelectrode array with dielectrophoresis (DEP). The relatively high sensitivity achieved with large electrode spacing can be attributed to the localization of fringing electric field lines through the trap holes occupied by bacterial analyte solution near the electrode. Using this technique an array of such sensitive sensors can be easily realized to yield a biochip for detection of various foodborne pathogens down to 10 3 CFU/ml.