Micromechanical oscillators as rapid biosensor for the detection of active growth of Escherichia coli

A rapid biosensor for the detection of bacterial growth was developed using micromechanical oscillators coated by common nutritive layers. The change in resonance frequency as a function of the increasing mass on a cantilever array forms the basis of the detection scheme. The sensor is able to detect active growth of Escherichia coli cells within 1 h which is significantly faster than any conventional plating method which requires at least 24 h. The growth of E. coli was confirmed by scanning electron microscopy. This new sensing method for the detection of active bacterial growth allows future applications in, e.g., rapid antibiotic susceptibility testing by adding antibiotics to the nutritive layer.     

Microcalorimetric study of the kinetics of the antibacterial effect of third generation cephalosporins in an in vitro dynamic system]

The antimicrobial effect (AME) kinetics of cefotaxime and ceftizoxime was studied with respect to 4 bacterial strains (E. coli, S. aureus, K. pneumoniae and P. aeruginosa) in an in vitro dynamic model. The mean integral concentrations, i.e. the AUC values divided by the dosing interval observed in blood of humans after the drug single intravenous administration in doses of 0.25, 0.5, 1 2 and 4 g. were simulated. The simulated concentrations of cefotaxime and ceftizoxime were 6.25 and 6.75, 12.5 and 13.5, 25 and 27, 50 and 54 and 100 and 108 mg/kg, respectively accounting for the differences in their pharmacokinetics. The changes in the microbial count were recorded microcalorimetrically by the rate of heat output with the BioActivity Monitor LKB 2277-202. The AME was expressed by TE reflecting the shift of the microbial growth curve in the presence of the cephalosporins against the control growth curve (in the absence of the drugs). It was noted that in simulating the concentrations observed after the drugs administration in various doses cefotaxime was mainly superior to ceftizoxime in terms of the TE, the value of the TE correlating with the respective values of the MICs. Some discrepancies between the TE and MICs were explained by the complicated shape of the TE vs. concentration or AUC curve observed earlier in regard to cefotaxime as well as by the differences in the steady state and dynamic conditions of the experiment.     

Synergistic effect of Myrtus communis L. essential oils and conventional antibiotics against multi-drug resistant Acinetobacter baumannii wound isolates

Acinetobacter baumannii is a rapidly emerging, highly resistant clinical pathogen with increasing prevalence. In recent years, the limited number of antimicrobial agents available for treatment of infections with multi-drug resistant (MDR) strains reinforced tendency for discovery of novel antimicrobial agents or treatment strategies. The aim of the study was to determine antimicrobial effectiveness of three Myrtus communis L. essential oils, both alone and in combination with conventional antibiotics, against MDR A. baumannii wound isolates. The results obtained highlighted the occurrence of good antibacterial effect of myrtle oils when administered alone. Using checkerboard method, the combinations of subinhibitory concentrations of myrtle essential oils and conventional antibiotics, i.e. polymixin B and ciprofloxacine were examined. The results proved synergism among M. communis L. essential oils and both antibiotics against MDR A. baumannii wound isolates, with a FIC index under or equal 0.50. Combination of subinhibitory concentrations of essential oils and ciprofloxacin most frequently reduced bacterial growth in synergistic manner. The similar has been shown for combination with polymyxin B; furthermore, the myrtle essential oil resulted in re-sensitization of the MDR wound isolates, i.e. MICs used in combination were below the cut off for the sensitivity to the antibiotic. Time-kill curve method confirmed efficacy of myrtle essential oil and polymyxin B combination, with complete reduction of bacterial count after 6 h. The detected synergy offers an opportunity for future development of treatment strategies for potentially lethal wound infections caused by MDR A. baumannii.     

A reagent less fluorescent sol-gel biosensor for uric acid detection in biological fluids

The simultaneous encapsulation of a coupled uricase-peroxidase system and amplex red in a sol-gel matrix allows one to obtain a reagent-less and ready-to-use fluorescent biosensor for the accurate detection of uric acid in highly diluted biological fluids. The detection limit of the prepared biosensor was found to be 20 nM and was linear up to 1 microM. The high sensitivity found for the biosensor permitted a reliable determination of uric acid concentrations in the presence of interfering species (e.g., ascorbic acid) just by sample dilution (up to 50000 for urine and 10000 for serum and blood). The sol-gel encapsulation preserved the hierarchy of the enzyme activity as demonstrated by the performance of the fluorescent biosensor.     

Design,synthesis and evaluation of carbazole derivatives as potential antimicrobial agents

Five series of novel carbazole derivatives containing an aminoguanidine, dihydrotriazine, thiosemicarbazide, semicarbazide or isonicotinic moiety were designed, synthesised and evaluated for their antimicrobial activities. Most of the compounds exhibited potent inhibitory activities towards different bacterial strains (including one multidrug-resistant clinical isolate) and one fungal strain with minimum inhibitory concentrations (MICs) between 0.5 and 16 µg/ml. Compounds 8f and 9d showed the most potent inhibitory activities (MICs of 0.5–2 µg/ml). Furthermore, compounds 8b, 8d, 8f, 8k, 9b and 9e with antimicrobial activities were not cytotoxic to human gastric cancer cell lines (SGC-7901 and AGS) or a normal human liver cell line (L-02). Structure–activity relationship analyses and docking studies implicated the dihydrotriazine group in increasing the antimicrobial potency and reducing the toxicity of the carbazole compounds. In vitro enzyme activity assays suggested that compound 8f binding to dihydrofolate reductase might account for the antimicrobial effect.     

Disposable bioluminescence-based biosensor for detection of bacterial count in food

A biosensor for rapid detection of bacterial count based on adenosine 5′-triphosphate (ATP) bioluminescence has been developed. The biosensor is composed of a key sensitive element and a photomultiplier tube used as a detector element. The disposable sensitive element consists of a sampler, a cartridge where intracellular ATP is chemically extracted from bacteria, and a microtube where the extracted ATP reacts with the luciferin–luciferase reagent to produce bioluminescence. The bioluminescence signal is transformed into relevant electrical signal by the detector and further measured with a homemade luminometer. Parameters affecting the amount of the extracted ATP, including the types of ATP extractants, the concentrations of ATP extractant, and the relevant neutralizing reagent, were optimized. Under the optimal experimental conditions, the biosensor showed a linear response to standard bacteria in a concentration range from 10³ to 10⁸ colony-forming units (CFU) per milliliter with a correlation coefficient of 0.925 (n = 22) within 5 min. Moreover, the bacterial count of real food samples obtained by the biosensor correlated well with those by the conventional plate count method. The proposed biosensor, with characteristics of low cost, easy operation, and fast response, provides potential application to rapid evaluation of bacterial contamination in the food industry, environment monitoring, and other fields.     

Sigma B对单核细胞增生李斯特菌耐受抗生素作用的影响

单核细胞增生李斯特菌是重要的革兰阳性食源性致病菌。近年来的报道显示出该菌耐受抗生素的能力有不断增强的趋势,为了探讨其耐药机制,对Sigma B（σB,李斯特菌中应对环境胁迫的主要调控因子）在抗生素耐受性中的作用进行了初步研究。检测和比较单核细胞增生李斯特菌标准菌株EGDe和其σB缺失突变菌株EGDeΔsigB对盘尼西林青霉素、氨苄西林青霉素、利福平、硫酸庆大霉素、四环素盐酸和红霉素6种抗生素的最小抑菌浓度（MIC）;在测定的MIC的基础上,利用MTT（噻唑蓝活体染色法）法比较EGDe和EGDeΔsigB在1×MIC、2×MIC和8×MIC的氨苄西林青霉素、红霉素和利福平3种抗生素中的生长活性。EGDe对盘尼西林青霉素（0.16μg/mL）、四环素盐酸（0.25μg/mL）和硫酸庆大霉素（0.5μg/mL）的MIC高于EGDeΔsigB（分别为0.08、0.125和0.125μg/mL）;而对氨苄西林青霉素、红霉素和利福平的MIC 2种菌株没有差别,分别为0.19、0.125和0.032μg/mL;与EGDe相比,EGDeΔsigB在氨苄西林青霉素、红霉素和利福平培养基中的生长活性较差,对抗生素的抑制更为敏感,而且随着这3种抗生素浓度的增加,其抑制程度也随之增强。Sigma B在单核细胞增生李斯特菌对抗生素的耐受中起到重要调节作用。     

Escherichia coli Cell Surface Perturbation and Disruption Induced by Antimicrobial Peptides BP100 and pepR

The potential of antimicrobial peptides (AMPs) as an alternative to conventional therapies is well recognized. Insights into the biological and biophysical properties of AMPs are thus key to understanding their mode of action. In this study, the mechanisms adopted by two AMPs in disrupting the Gram-negative Escherichia coli bacterial envelope were explored. BP100 is a short cecropin A-melittin hybrid peptide known to inhibit the growth of phytopathogenic Gram-negative bacteria. pepR, on the other hand, is a novel AMP derived from the dengue virus capsid protein. Both BP100 and pepR were found to inhibit the growth of E. coli at micromolar concentrations. Zeta potential measurements of E. coli incubated with increasing peptide concentrations allowed for the establishment of a correlation between the minimal inhibitory concentration (MIC) of each AMP and membrane surface charge neutralization. While a neutralization-mediated killing mechanism adopted by either AMP is not necessarily implied, the hypothesis that surface neutralization occurs close to MIC values was confirmed. Atomic force microscopy (AFM) was then employed to visualize the structural effect of the interaction of each AMP with the E. coli cell envelope. At their MICs, BP100 and pepR progressively destroyed the bacterial envelope, with extensive damage already occurring 2 h after peptide addition to the bacteria. A similar effect was observed for each AMP in the concentration-dependent studies. At peptide concentrations below MIC values, only minor disruptions of the bacterial surface occurred.     

A New,Sensitive Marine Microalgal Recombinant Biosensor Using Luminescence Monitoring for Toxicity Testing of Antifouling Biocides

In this study, we propose the use of the marine green alga Ostreococcus tauri, the smallest free-living eukaryotic cell known to date, as a new luminescent biosensor for toxicity testing in the environment. Diuron and Irgarol 1051, two antifouling biocides commonly encountered in coastal waters, were chosen to test this new biosensor along with two degradation products of diuron. The effects of various concentrations of the antifoulants on four genetic constructs of O. tauri (based on genes involved in photosynthesis, cell cycle, and circadian clock) were compared using 96-well culture microplates and a luminometer to automatically measure luminescence over 3 days. This was compared to growth inhibition of O. tauri wild type under the same conditions. Luminescence appeared to be more sensitive than growth inhibition as an indicator of toxicity. Cyclin-dependent kinase (CDKA), a protein involved in the cell cycle, fused to luciferase (CDKA-Luc) was found to be the most sensitive of the biosensors, allowing an accurate determination of the 50% effective concentration (EC₅₀) after only 2 days (diuron, 5.65 ± 0.44 μg/liter; Irgarol 1015, 0.76 ± 0.10 μg/liter). The effects of the antifoulants on the CDKA-Luc biosensor were then compared to growth inhibition in natural marine phytoplankton. The effective concentrations of diuron and Irgarol 1051 were found to be similar, indicating that this biosensor would be suitable as a reliable ecotoxicological test. The advantage of this biosensor over cell growth inhibition testing is that the process can be easily automated and could provide a high-throughput laboratory approach to perform short-term toxicity tests. The ability to genetically transform and culture recombinant O. tauri gives it huge potential for screening many other toxic compounds.     

A multipurpose capacitive biosensor for assay and quality control of human immunoglobulin G

We report a flow‐injection biosensor system with a capacitive transducer for assay and quality control of human immunoglobulin G (hIgG). The sensing platform is based on self‐assembled monolayers (SAMs) of carboxylic acid terminated alkyl‐thiols with covalently attached concanavalin A. The electrochemical characteristics of the sensor surface were assessed by cyclic voltammetry using a permeable redox couple (potassium ferricyanide). The developed biosensor proved capable of performing a sensitive label‐free assay of hIgG with a detection limit of 1.0 µg mL^?1. The capacitance response depended linearly on hIgG concentration over the range from 5.0 to 100 µg mL^?1, in a logarithmic plot. Typical measurements were performed in 15 min and up to 18 successive assays were achieved without significant loss of sensitivity using a single electrode. In addition, the biosensor can detect hIgG aggregates with concentrations as low as 0.01% of the total hIgG content (5.0 µg mL^?1). Hence, it represents a potential post‐size‐exclusion chromatography–UV (post‐SEC–UV) binding assay for in‐process quality control of hIgG, which cannot be detected by SEC–UV singly at concentrations below 0.3% of the total hIgG content. Biotechnol. Bioeng. 2009; 104: 312–320 © 2009 Wiley Periodicals, Inc.     

Benzene analysis in workplace air using an FIA-based bacterial biosensor

A bacterial biosensor based on flow injection analysis (FIA) has been developed for the determination of benzene in workplace air samples. Benzene can be used by the bacteria Pseudomonas putida ML2 as a sole carbon source, and its aerobic degradation can be measured using a dissolved oxygen electrode. The bacterial cells were immobilised between two cellulose acetate membranes and fixed onto a Clark dissolved oxygen probe, which was inserted into a custom-made flow cell. The applicability of the biosensor for the analysis of air samples containing benzene was investigated. Air samples were collected from a controlled exposure room using charcoal adsorption tubes, and benzene extracted with solvent desorption using dimethylformamide (DMF). The biosensor displayed a linear detection range between 0.025 and 0.15 mM benzene based on standard solutions containing a maximum of 2% DMF, with a response time of 6 min. This linear detection range allows the analysis of air containing between 3 and 16 ppm benzene based on a 60-min sampling period. DMF proved to be compatible for use with the biosensor, causing minimal interference with the sensor response and causing no toxic effects on the bacterial cells. The FIA system was easily transported to an in situ location, and a correlation was obtained between the biosensor and gas chromatography (GC) results for the preliminary air samples investigated. Moreover, the biosensor displayed no interference to other benzene related compounds in the BTEX range. The results from this work have shown that the biosensor has potential applications for the analysis of benzene in workplace air samples, with the added advantages over the conventional GC methods of low operation costs, ease of use, and portability for in situ measurements.     

Sub-Minimum Inhibitory Concentrations of Ceftibuten Reduce Adherence of Escherichia coli to Human Cells and Induces Formation of Long Filaments

The minimal inhibitory concentrations (MICs) of an antibiotic are present for only a certain period of time, after which they become sub-inhibitory concentrations (sub-MICs). These sub-MICs are still active because they can interfere with the mechanism of bacterial adhesion, which is the first step in the sequence of events leading to infection. The purpose of the present study was to investigate the effects of sub-MICs of ceftibuten, a new third-generation cephalosporin, on the adhesion of Escherichia coli (E. coli) to human buccal cells. The degree of inhibition was maximal at 1/2 MIC and then gradually returned toward to the control values at 1/128 the MIC. The differences were statistically significant from 1/2 to 1/32 MIC. Since the MIC was 0.5 μg/ml, concentrations from 0.25 to 0.015 μg/ml significantly reduce bacterial adhesion. Ceftibuten also caused marked elongation of E. coli. These findings could help to explain the efficacy showed by ceftibuten in the treatment of respiratory and urinary tract infections when administered once daily.     

Rapid biosensor for detection of antibiotic-selective growth of Escherichia coli

A rapid biosensor for the detection of bacterial growth was developed using micromechanical oscillators coated in common nutritive layers. The change in resonance frequency as a function of the increasing mass on a cantilever array forms the basis of the detection scheme. The calculated mass sensitivity according to the mechanical properties of the cantilever sensor is approximately 50 pg/Hz; this mass corresponds to an approximate sensitivity of approximately 100 Escherichia coli cells. The sensor is able to detect active growth of E. coli cells within 1 h. The starting number of E. coli cells initially attached to the sensor cantilever was, on average, approximately 1,000 cells. Furthermore, this method allows the detection of selective growth of E. coli within only 2 h by adding antibiotics to the nutritive layers. The growth of E. coli was confirmed by scanning electron microscopy. This new sensing method for the detection of selective bacterial growth allows future applications in, e.g., rapid antibiotic susceptibility testing.     

Development of a whole cell green fluorescent sensor for lysine quantification

As an essential amino acid, lysine is an important component of animal and human diets and its bioavailability can depend on a variety of factors. Therefore, an accurate pre-determination of bioavailable lysine in foods and feeds is important. In this study a whole cell fluorescent biosensor for the quantification of lysine in protein sources was constructed. A gene encoding for green fluorescent protein (GFPmut3) was introduced into an E. coli lysine auxotroph genome as a part of a mini-Tn5-Km transposon. The location of the transposon was determined and the growth kinetics of the newly constructed biosensor were examined. The transposon disrupted the ybhM gene, which encodes for the synthesis of a protein with an unknown function. No effect of the transposon’s location in the genome or the expression of gfp on bacterial growth rates was observed. Based on the fluorescence emitted by GFPmut3, a standard curve after 6-h growth of the strain was generated. A correlation coefficient of 0.95 was observed when the fluorescence method was compared to the conventional optical density (OD) growth-based lysine assay. Using the newly developed lysine fluorescent whole cell sensor we determined the total lysine in casein acid hydrolyzate (7.13 ± 0.34%). When lysine added to 12 μg/ml and 30 μg/ml of casein acid hydrolyzate was quantified, recoveries of 97 ± 1.65% and 103 ± 4.66% respectively were detected. The results suggest that the microbial assay using GFP fluorescence represents a promising alternative method for the potential estimation of lysine in protein sources.     

Localized surface plasmon coupled fluorescence fiber-optic biosensor for alpha-fetoprotein detection in human serum

In this study, we demonstrated that the fiber-optic biosensor based on localized surface plasmon coupled fluorescence (LSPCF) is capable of detecting alpha-fetoprotein (AFP) in human serum. The sensitivity of LSPCF fiber-optic biosensor is not only enhanced but also the specific selectivity is improved since the fluorophores are excited by the localized surface plasmon with high efficiency. Experimentally, this fiber-optic biosensor is able to detect AFP concentration in phosphate buffered saline (PBS) solution from 0.1ng/mL to 100ng/mL whereas the linear relationship between the AFP concentrations and the fluorescence signals is shown. Furthermore, a linear response between the fluorescence signals and the concentrations of AFP in human serum from 2.33ng/mL to 143.74ng/mL is also obtained. As a result, the detection limit of the LSPCF fiber-optic biosensor on AFP detection is comparable with the conventional enzyme-linked immunosorbent assay (ELISA). Additionally, the LSPCF fiber-optic biosensor benefits on inexpensive, disposable and simpler optical geometry that can become a high efficient immunoassay comparable with the conventional ELISA and radioimmunoassay (RIA) clinically.     

Semi-quantitative detection of gene expression using bisbenzimide dye

An electrochemical biosensor, using a disposable electrochemical printed chip aggregation by the bisbenzimide dye (Hoechst 33258), was used for detecting the expression of β-actin and RAGE genes. Using linear sweep voltammetry, the expression of these two genes in HeLa and HepG2 cell lines was determined based on anodic peak current, and the results were compared with conventional agarose gel electrophoresis. Total cellular RNA was reverse transcribed to complementary DNA, and amplification by PCR was carried out. Subsequently, the PCR products were subjected to detection by either electrophoresis or electrochemical biosensor. Precision of the electrochemical biosensor technique was acceptable (β-actin: CV = 1.875% for 10(4) copies and 4.684% for 10(9) copies; RAGE: CV = 2.253% for 10(9) copies, and 3.743% for 10 copies). In the electrochemical biosensor technique, the PCR products were measured in the same run with various concentrations of standards, and copy numbers of β-actin gene were interpolated from a standard curve. Copy numbers of the β-actin gene were then compared between the two techniques. At the 95% confidence limit, the two methods had no significant differences and were significantly correlated (y = -40383.0623 + 1.0233x; P > 0.10). The electrochemical biosensor method was more sensitive than the conventional electrophoresis method because it could detect as low as 10 copies of the RAGE gene. The conventional electrophoresis method detected the RAGE gene at concentrations of at least 10(4) copies, and the linearity for semi-quantitative measurement was in the range of 10(6)-10(9) copies. When the electrochemical biosensor was applied to detect the RAGE gene expression in both cell types, we found that HeLa cells expressed the RAGE gene about 2-fold higher than in HepG2 cells (relative value of 0.000905 vs 0.0004670). Therefore, this study suggests the potential modification of the electrochemical biosensor with the use of bisbenzimide dye (Hoechst 33258) for detecting gene expression.     

Isolation and characterization of heavy-metal resistant microbes from roadside soil and phylloplane

Contamination by heavy metals is one of the major environmental problems in many countries and these contaminants reach from various sources such as traffic cars and other activities. Soil and phylloplane samples were collected from eight traffic and two non-traffic sites in Sohag city, Egypt. Heavy metal contents of Cd2?, Zn2? and Pb2? of soil and phylloplane samples were determined and revealed high levels of Zn2? and Pb2? in traffic samples. A total of 112 bacterial and 62 fungal isolates were obtained from soil and phylloplane. Bacterial isolates were characterized on the basis of morphological, physicochemical and biochemical characteristics; and 16S rRNA gene sequences. Fungal isolates were identified according to morphological characterization. Minimal inhibitory concentrations (MICs) of Cd2?, Zn2? and Pb2? for each isolate were detected. All bacterial and fungal isolates demonstrated resistance to lead with MICs >0.528 mM and >0.211, respectively. Moreover, the maximum MICs of cadmium and zinc for bacteria were 0.821 mM and 1.471 mM, respectively, where as, MICs for fungi were 0.328 mM and 0.588 mM, respectively. The most resistant bacterial and fungal isolates were Pseudomonas aeruginosa RA65 and Penicillium corylophyllum, respectively. Therefore, P. aeruginosa RA65 was selected for further investigations. Growth curve study showed that 0.264 mM lead had no efficiently effect on the growth of P. aeruginosa RA65. Plasmid isolation evidenced by transformation studies indicated that P. aeruginosa RA65 harbored a single plasmid (~9.5 kb) which mediated heavy meal resistance. Consequently, these microbial isolates could be potentially used in bioremediation of heavy metal-contaminated environment.     

Application of an Escherichia coli green fluorescent protein-based lysine biosensor under nonsterile conditions and autofluorescence background

AIMS: To examine the utility of an Escherichia coli green fluorescent protein (GFP) containing biosensor for quantification of bioavailable lysine in selected feed samples under nonsterile conditions and to estimate the background fluorescence of analyzed feed samples and evaluate the risk of confounding GFP emission from the lysine assay organism. METHODS AND RESULTS: Escherichia coli lysine auxotroph GFP based biosensor was used to determine the percentage of bioavailable lysine in two samples of soybean-, cottonseed-, and meat and bone meal under nonsterile conditions. The fluorescence emitted by GFP was successfully measured using a spectrofluorimeter to monitor bacterial growth response to protein-derived lysine and lysine containing small peptides. The autofluorescence of analyzed feed samples at different concentrations could also be estimated. CONCLUSIONS: When feed protein concentrations are decreased, autofluorescence interference can be avoided. SIGNIFICANCE: The E. coli lysine auxotroph GFP-based biosensor can successfully be used for the determination of bioavailable lysine in these selected animal feed proteins under nonsterile conditions. IMPACT OF THE STUDY: E. coli GFP biosensor for lysine has potential for routine application in animal feeds.     

Inhibition of the Metabolism of Streptococci and Salmonella by Specific Antisera

Streptococcal and salmonella antisera inhibited carbohydrate metabolism for groups A, B, C, and D streptococci and group E salmonella, as measured by the formation of [(14)C]dioxide from [(14)C]glucose metabolism. For salmonella, the inhibition was type specific since group E salmonella were inhibited only by salmonella E antisera and not by anti-salmonella A or C(1). For streptococci, quantitative differences were demonstrated, but major cross-reactivity was observed. At high concentrations, the antisera were bactericidal; at more dilute concentrations, for both salmonella and streptococci, carbohydrate metabolism was suppressed, but subculture on chocolate agar showed abundant growth. Cross-reacting antibodies could be absorbed by incubation with either antigen, e.g., streptococcal antisera versus heat-killed salmonella. The results suggest that the radiometric technique can be more sensitive than either capillary flocculation or visual detection of bacterial growth for detecting the inhibition of streptococci and salmonella by specific antibodies. The use of specific antisera may prove useful for bacterial species identification in an automated system for detection of bacterial growth.