Method for assessment of functional affinity of antibodies for live bacteria

A rapid and convenient method for assessment of functional affinity of antibodies against live bacteria is described. When a combination of immunomagnetic separation (IMS) with bioluminescent or fluorescent genetic labelling of the cells was employed, the method showed good correlation with plate count. However, the use of reporter bacteria allowed results to be obtained within 1 h compared with days using conventional methods. Due to its lower detection limit, the bioluminescent assay performed better than the fluorescent assay. Antibody affinities for Escherichia coli O157:H7 and Salmonella enteritidis were examined at different environmental conditions such as pH 3-7, temperature 4-25 degrees C, and sodium chloride concentrations 0-5% and compared with sensitivities of ELISA.     

Measurement of bacterial adhesion-in vitro evaluation of different methods

The adhesion of bacteria to host tissue is the first step in pathogenesis. Similarly, bacterial adhesion to inanimate surfaces is the first step in formation of biofilms-a real problem in industrial processes and medical devices. Various agents capable of blocking the adhesion of bacteria to surfaces have been identified, such as probiotics, which are supposed to prevent the adhesion of pathogenic bacteria to the intestinal mucosa. Although measurement of bacterial adhesion is important itself, especially when agents used to prevent adhesion are developed, a relative small number of techniques can be used in the measurement of adhesion. These techniques are not well validated and there is lack of studies where those methods are compared to each other. Here we have compared different commonly used methods to measure adhesion of bacteria; radioactive labelling, fluorescence tagging, and staining of bacteria. The methods were used to measure the adhesion of Escherichia coli and Salmonella enterica serovar Typhimurium to intestinal mucus. Moreover, selected probiotic strains were used to study whether probiotics or the adhesion method used affected the results. As a result, we show that the best reproducibility and sensitivity were obtained using radioactive labelling. With other methods, the sensitivity was too low due to poorly adhering bacteria and low signal-to-background ratio.     

Production and characterization of anti-bifidobacteria monoclonal antibodies and their application in the development of an immuno-culture detection method

An immuno-culture method has been developed by combination of specific monoclonal antibodies and plate culture to allow detection of viable bifidobacteria. Cell wall proteins were selected as surface antigen to produce antibodies against bifidobacteria. The cell wall proteins were extracted and purified from six ATCC strains of bifidobacteria grown in MRS broth using an anaerobic system. To compare the profile of the protein extracts, all the protein solutions obtained were analyzed by SDS-PAGE. Similar bands corresponding to the major proteins of each species of bifidobacteria were observed. The proteins were tested for their immunogenicity in Balb/c mice after immunization and subsequent analysis using ELISA procedures. High immune responses were generated in mice immunized by proteins from Bifidobacterium bifidum and Bifidobacterium longum. Monoclonal antibodies were produced against B. longum and tested for their specificity, sensitivity and cross reactivity with other bifidobacteria species. All the hybridoma cells selected produced anti-B. longum antibodies cross-reacting with native and purified proteins from five other bifidobacteria species. An epitope supported by a cross-reacting protein of 58 kDa shared by bifidobacteria was revealed by western blot. This was confirmed by immune-transmission electron microscopy observations which showed the specific interaction of these antibodies with bifidobacterial cell wall proteins. Also, the antibody obtained was found to be specific for the genus Bifidobacterium and sensitive, allowing the detection of at least 10(5) target cells/ml. An immuno-culture detection approach was then developed using the selected anti-B. longum antibodies. This method was shown to be very efficient for the detection of viable cells of bifidobacteria suggesting the possibility of its use to quantify these bacteria in various food matrices.     

Evaluation of the methods for enumerating coliform bacteria from water samples using precise reference standards

AIMS: To use BioBall cultures as a precise reference standard to evaluate methods for enumeration of Escherichia coli and other coliform bacteria in water samples. METHODS AND RESULTS: Eight methods were evaluated including membrane filtration, standard plate count (pour and spread plate methods), defined substrate technology methods (Colilert and Colisure), the most probable number method and the Petrifilm disposable plate method. Escherichia coli and Enterobacter aerogenes BioBall cultures containing 30 organisms each were used. All tests were performed using 10 replicates. The mean recovery of both bacteria varied with the different methods employed. CONCLUSIONS: The best and most consistent results were obtained with Petrifilm and the pour plate method. Other methods either yielded a low recovery or showed significantly high variability between replicates. SIGNIFICANCE AND IMPACT OF THE STUDY: The BioBall is a very suitable quality control tool for evaluating the efficiency of methods for bacterial enumeration in water samples.     

Antibacterial activity and mechanism of action of the silver ion in Staphylococcus aureus and Escherichia coli

The antibacterial effect and mechanism of action of a silver ion solution that was electrically generated were investigated for Staphylococcus aureus and Escherichia coli by analyzing the growth, morphology, and ultrastructure of the bacterial cells following treatment with the silver ion solution. Bacteria were exposed to the silver ion solution for various lengths of time, and the antibacterial effect of the solution was tested using the conventional plate count method and flow cytometric (FC) analysis. Reductions of more than 5 log(10) CFU/ml of both S. aureus and E. coli bacteria were confirmed after 90 min of treatment with the silver ion solution. Significant reduction of S. aureus and E. coli cells was also observed by FC analysis; however, the reduction rate determined by FC analysis was less than that determined by the conventional plate count method. These differences may be attributed to the presence of bacteria in an active but nonculturable (ABNC) state after treatment with the silver ion solution. Transmission electron microscopy showed considerable changes in the bacterial cell membranes upon silver ion treatment, which might be the cause or consequence of cell death. In conclusion, the results of the present study suggest that silver ions may cause S. aureus and E. coli bacteria to reach an ABNC state and eventually die.     

Demonstration of nitric oxide synthase activity in crustacean hemocytes and anti-microbial activity of hemocyte-derived nitric oxide

We determined the biochemical characteristics of nitric oxide synthase (NOS) in hemocytes of the crayfish Procambarus clarkii and investigated the roles of hemocyte-derived NO in host defense. Biochemical analysis indicated the presence of a Ca2+ -independent NOS activity, which was elevated by lipopolysaccharide (LPS) treatment. When bacteria (Staphylococcus aureus) and hemocytes were co-incubated, adhesion of bacteria to hemocytes was observed. NO donor sodium nitroprusside (SNP) significantly increased the numbers of hemocytes to which bacteria adhered. Similarly, LPS elicited bacterial adhesion and the LPS-induced adhesion was prevented by NOS inhibitor NG-monomethyl-L-arginine (L-NMMA). Finally, plate count assay demonstrated that addition of LPS to the hemocytes/bacteria co-incubation resulted in a significant decrease in bacterial colony forming unit (CFU), and that L-NMMA reversed the decreasing effect of LPS on CFU. The combined results demonstrate the presence of a Ca2+ -independent LPS-inducible NOS activity in crayfish hemocytes and suggest that hemocyte-derived NO is involved in promoting bacterial adhesion to hemocytes and enhancing bactericidal activity of hemocytes.     

New method to study bacterial adhesion to meat.

A new method was developed for the study of bacterial adhesion to meat surfaces. Thin slices of meat (40 microns thick) were inserted into a specially designed observation chamber. The meat slices were then exposed to a bacterial suspension (ca. 10(6) CFU.ml-1) to initiate adhesion (20 min of contact time) and subsequently rinsed to eliminate nonadherent bacteria. Because of the special chamber design, the disruptive force exerted on the bacteria during rinsing (shear stress) was uniform over the whole surface of the meat slices, was constant, and could be varied from 0 to 0.08 N.m-2. After being rinsed, the meat slices were stained with basic fuschin and observed under light microscopy to determine the number and distribution of adherent bacteria. This new method was used to study the adhesion of Acinetobacter strain LD2, a Lactobacillus sp., and Pseudomonas fluorescens to slices of beef fat and tendon. At 25 degrees C, most (greater than or equal to 99.9%) of the cells of the Lactobacillus sp. deposited on the meat were washed off the surface during rinsing (0.05 N.m-2), whereas a large number (ca. 10(5) CFU.cm-2) of Acinetobacter strain LD2 and P. fluorescens cells remained adherent. The extent of adhesion was similar on fat and tendon, and adherent bacteria were distributed evenly over the whole surface of the slices. This preliminary study indicates that the combined use of thin slices of meat and of the observation chamber provides us with the means to more accurately study bacterial adhesion to meat surfaces.     

New method to study bacterial adhesion to meat

A new method was developed for the study of bacterial adhesion to meat surfaces. Thin slices of meat (40 microns thick) were inserted into a specially designed observation chamber. The meat slices were then exposed to a bacterial suspension (ca. 10(6) CFU.ml-1) to initiate adhesion (20 min of contact time) and subsequently rinsed to eliminate nonadherent bacteria. Because of the special chamber design, the disruptive force exerted on the bacteria during rinsing (shear stress) was uniform over the whole surface of the meat slices, was constant, and could be varied from 0 to 0.08 N.m-2. After being rinsed, the meat slices were stained with basic fuschin and observed under light microscopy to determine the number and distribution of adherent bacteria. This new method was used to study the adhesion of Acinetobacter strain LD2, a Lactobacillus sp., and Pseudomonas fluorescens to slices of beef fat and tendon. At 25 degrees C, most (greater than or equal to 99.9%) of the cells of the Lactobacillus sp. deposited on the meat were washed off the surface during rinsing (0.05 N.m-2), whereas a large number (ca. 10(5) CFU.cm-2) of Acinetobacter strain LD2 and P. fluorescens cells remained adherent. The extent of adhesion was similar on fat and tendon, and adherent bacteria were distributed evenly over the whole surface of the slices. This preliminary study indicates that the combined use of thin slices of meat and of the observation chamber provides us with the means to more accurately study bacterial adhesion to meat surfaces.     

Enzymic detection of adhesion of enteropathogenic Escherichia coli to HEp-2 cells

We established a new method for detecting enteropathogenic Escherichia coli adhering to HEp-2 cells. An essential part of the method is an assay of beta-galactosidase activity of adhered bacterial cells. It consisted of the following steps: (1) culture of bacterial cells in a medium containing isopropyl-thio-beta-D-galactoside, an inducer of beta-galactosidase; (2) incubation of a bacterial culture with monolayered HEp-2 cells in a 96-well culture plate; (3) washing wells to remove bacterial cells which did not adhere to HEp-2 cells, and (4) enzymic reaction for beta-galactosidase activities. However, a calibration curve for the enzyme activity, obtained from each bacterial sample, showed that 10(5) bacteria per well permitted an accurate estimation. The enzyme activity of adhered bacteria to the monolayered cells showed that 10(7) bacteria were appropriate for the adherence assay. The number of adhered bacteria thus obtained was in good agreement with a viable cell count. The result indicates that the new method is more reliable than a widely used method, counting the number of bacteria under a microscope. The present method also makes it easy to detect adherent strains of E. coli in large numbers of specimens.     

Development of antibody array for simultaneous detection of foodborne pathogens

Pathogenic bacterial contaminations present serious problems for food industry and public health. Rapid, accurate and affordable assays are needed. In this study, antibody arrays to simultaneously detect two foodborne pathogenic bacteria (Escherichia coli O157:H7 and Salmonella spp.) have been developed using chemiluminescent detecting system. Solid supports using nitrocellulose membrane and poly-l-lysine (PLL) glass slide were compared and optimized for antibody array construction. Many parameters including optimal concentrations of antibodies, blocking reagents, assay time, storage time, sensitivity and cross-reactivity were considered during optimization. This study revealed that the PLL slide was a more suitable support due to highly accurate results and the absence of non-specific background. Phosphate-buffered saline (PBS, pH 7.2) and 3% skim milk in PBS buffer were optimal spotting and blocking reagents, respectively. With the same sensitivity for bacterial detection as in a conventional ELISA (10(5)-10(6)CFU/ml for the E. coli O157:H7 and 10(6)-10(7)CFU/ml for Salmonella detections), this antibody array has advantages of a much shorter assay time of 1h and much lower required amounts of antibodies. Moreover, there was no cross-reactivity in the detection among bacteria tested in this study. Bacteria detection in food sample was feasible as demonstrated using bacteria-added milk.     

不同方法检测鸡蛋表面菌落总数的相关性研究

研究了ATP生物荧光检测法与国标法《GB4789.2-2010食品卫生微生物学检验菌落总数测定》检测鸡蛋壳表面细菌总数的相关性。采用ATP生物荧光检测法和国标法对40个混合样品表面细菌总数进行检测,以log CFU/个蛋壳为横坐标(x),以logRLU/个蛋壳为纵坐标(y),分别进行线性、对数、乘幂、指数拟合。结果表明,ATP荧光检测法与国标法检测结果 Pearson相关系数为0.912,线性模型y=0.7306x-1.0041(R2=0.8322)拟合度较高。该试验结果为ATP荧光检测法在鸡蛋壳表面细菌总数快速检测中应用的可行性提供了依据。     

A robust and fast bacteria counting method using CdSe/ZnS core/shell quantum dots as labels

A rapid, sensitive fluorescence measurement method for detecting the bacterial count using CdSe/ZnS as a fluorescence marker was described. High-quality CdSe/ZnS nanocrystals were synthesized and successfully conjugated with bacteria. The fluorescence intensity was proportional to bacterial count in the range of 10²–10⁸ CFU/mL and the low detection limit was 10² CFU/mL.     

ESTIMATION OF MICROBIOLOGICAL QUALITY OF CHILLED BEEF USING AUTOMATED TURBIDIMETRY

Total bacterial counts on chilled beef samples were estimated by the standard plate count method and by an automated turbidimetric system. The latter method is based on product-specific calibration curves constructed by correlating growth curve parameters calculated for the turbidimeter to the log CFU values obtained by plate counts. A total of 74 beef samples was used to construct the calibration curves. Correlation analysis between turbidimetric parameters and plate count values showed that detection time was the best predictor to estimate microbial loads on fresh (r=0.91) and aged beef (r=0.94). Microbial loads for a different set of aged beef samples (n = 37) refrigerated for 7, 9, 10, 17 and 45 days were compared by turbidimetric measurements and plate counts. Mean total viable counts were log 5.92 ± 1.17 and log 5.54 ± 1.28 CFU/mL, respectively. Results showed that total bacterial counts on chilled beef could be estimated accurately from turbidimetric parameters. Furthermore, setting a cut-off value of log 6 CFU/mL allowed to accepting/rejecting samples according to their microbial condition in shorter periods of time compared to the traditional plate count method.     

Determination of the viability of Aeromonas hydrophila in different types of water by flow cytometry, and comparison with classical methods

The presence of Aeromonas spp. in water can represent a risk for human health. Therefore, it is important to know the physiological status of these bacteria and their survival in the environment. We studied the behavior of a strain of Aeromonas hydrophila in river water, spring water, brackish water, mineral water, and chlorinated drinking water, which had different physical and chemical characteristics. The bacterial content was evaluated by spectrophotometric and plate count techniques. Flow cytometric determination of viability was carried out using a dual-staining technique that enabled us to distinguish viable bacteria from damaged and membrane-compromised bacteria. The traditional methods showed that the bacterial content was variable and dependent on the type of water. The results obtained from the plate count analysis correlated with the absorbance data. In contrast, the flow cytometric analysis results did not correlate with the results obtained by traditional methods; in fact, this technique showed that there were viable cells even when the optical density was low or no longer detectable and there was no plate count value. According to our results, flow cytometry is a suitable method for assessing the viability of bacteria in water samples. Furthermore, it permits fast detection of bacteria that are in a viable but nonculturable state, which are not detectable by conventional methods.     

Quantitative approach in the study of adhesion of lactic acid bacteria to intestinal cells and their competition with enterobacteria

To describe the phenomena of bacterial adhesion to intestinal cells and the competition for adhesion between bacteria, mathematical equations based on a simple dissociation process involving a finite number of bacterial receptors on intestinal cell surface were developed. The equations allow the estimation of the maximum number of Lactobacillus sp. and Escherichia coli cells that can adhere to Caco-2 cells and intestinal mucus; they also characterize the affinity of the bacteria to Caco-2 cells and intestinal and fecal mucus and the theoretical adhesion ratio of two bacteria present in a mixed suspension. The competition for adhesion between Lactobacillus rhamnosus GG and E. coli TG1 appeared to follow the proposed kinetics, whereas the competition between Lactobacillus casei Shirota and E. coli TG1 may involve multiple adhesion sites or a soluble factor in the culture medium of the former. The displacement of the adhered Lactobacillus by E. coli TG1 seemed to be a rapid process, whereas the displacement of E. coli TG1 by the Lactobacillus took more than an hour.     

Impedance microbiology: quantification of bacterial content in milk by means of capacitance growth curves

The impedancimetric method is a technique for the rapid evaluation of milk bacterial content and also of its subproducts. Several authors have made use of culture conductance changes during bacterial growth for quantitative and qualitative assessments of microbial growth. However, interface capacitance curves, Ci, have not been used. In this paper, we quantify bacteria in cow raw milk by following their growth as the above-mentioned capacitance change time course event. With it, bigger growth variations, shorter detection times and a better coefficient of correlation with the plate count method were obtained than those yielded by conductance curves. Calibration was performed by plotting initial known concentrations, IC (CFU/ml), as a function of the time detection theshold (TDT).     

Use of impedance test for testing effectiveness of drug preservatives

Pharmacopoeias contain preservation efficacy test for estimating antimicrobial activity of chemical compounds added to pharmaceutical preparations in multidose containers in order to inhibit bioburden growth. This method involves the treating of preserved products with bacteria and yeast cells and monitoring the survival of microorganisms through the specified time periods up to 28 days. The last stage of assay--incubation and colony counting--is very time consuming. Recent advance in technology enables faster and more convenient detection in comparison to traditional methods. Impedance method is based on the principle that conductance and capacitance of cultivation medium increases when bacteria grow and metabolize, Impedance time detection is inversely proportional to initial bacterial population. Six different products were utilised throughout the study. The calibration curves were calculated for each of the tested strains by comparison between standard plate count method and detection time measured in Bactometer system. In our study log reduction calculated in alternative method were similar to those obtained in plate count assay. All of the tested preparations, except one, exhibited acceptable activity against bacteria and fungi and meet the pharmacopoeal requirements. The studies indicated a positive correlation between standard plate count results and impedance reading. The procedure with the usage of Bactometer, provides a rapid and accurate system for the determination of bacterial content.     

Determination of the Viability of Aeromonas hydrophila in Different Types of Water by Flow Cytometry, and Comparison with Classical Methods

The presence of Aeromonas spp. in water can represent a risk for human health. Therefore, it is important to know the physiological status of these bacteria and their survival in the environment. We studied the behavior of a strain of Aeromonas hydrophila in river water, spring water, brackish water, mineral water, and chlorinated drinking water, which had different physical and chemical characteristics. The bacterial content was evaluated by spectrophotometric and plate count techniques. Flow cytometric determination of viability was carried out using a dual-staining technique that enabled us to distinguish viable bacteria from damaged and membrane-compromised bacteria. The traditional methods showed that the bacterial content was variable and dependent on the type of water. The results obtained from the plate count analysis correlated with the absorbance data. In contrast, the flow cytometric analysis results did not correlate with the results obtained by traditional methods; in fact, this technique showed that there were viable cells even when the optical density was low or no longer detectable and there was no plate count value. According to our results, flow cytometry is a suitable method for assessing the viability of bacteria in water samples. Furthermore, it permits fast detection of bacteria that are in a viable but nonculturable state, which are not detectable by conventional methods.     

Adhesion of a Staphylococcus aureus strain to biomaterials does not select methicillin-resistant mutants

Bacterial adhesion to polymethylmethacrylate and to silicon elastomer, materials frequently used in clinical applications, has been investigated to assess whether adhesion selects methicillin-resistant mutants in the bacterial population in contact with the materials. The methicillin susceptibility of a susceptible Staphylococcus aureus (ATCC 25923) was measured by a modification of plate antibiogram Kirby-Bauer method, which allows optimised detection of small variations in antibiotic susceptibility. In both adherent and non-adherent bacterial subpopulations, the presence of mecA gene, which encodes for the protein PBP 2a responsible for methicillin resistance was searched for by Polymerase Chain Reaction (PCR). The contact with the two polymers did not induce in the bacteria population any phenotypic increase in methicillin resistance, or the selection of mutants carrying the mecA gene.     

A self-assembled monolayer-based piezoelectric immunosensor for rapid detection of Escherichia coli O157:H7

A piezoelectric immunosensor was developed for rapid detection of Escherichia coli O157:H7. It was based on the immobilization of affinity-purified antibodies onto a monolayer of 16-mercaptohexadecanoic acid (MHDA), a long-chain carboxylic acid-terminating alkanethiol, self-assembled on an AT-cut quartz crystal's Au electrode surface with N-hydroxysuccinimide (NHS) ester as a reactive intermediate. The binding of target bacteria onto the immobilized antibodies decreased the sensor's resonant frequency, and the frequency shift was correlated to the bacterial concentration. The stepwise assembly of the immunosensor was characterized by means of both quartz crystal microbalance (QCM) and cyclic voltammetry techniques. Three analytical procedures, namely immersion, dip-and-dry and flow-through methods, were investigated. The immunosensor could detect the target bacteria in a range of 10(3)-10(8)CFU/ml within 30-50 min, and the sensor-to-sensor reproducibility obtained at 10(3) and 10(5) colony-forming units (CFU)/ml was 18 and 11% R.S.D., respectively. The proposed sensor was comparable to Protein A-based piezoelectric immunosensor in terms of the amount of immobilized antibodies and detection sensitivity.