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Microbiological studies were performed on ten samples from a water body in a metropolitan district with a view to evaluating its epidemiological safety. The amounts of microorganisms from the main groups that include pathogenic species, were determined using microscopical, cultural and biochemical methods. The quantities of Gram-negative aerobic and facultative-anaerobic bacteria (5.50x10²±6.43 - 5.74x10²±8.87 CFU/ml) and staphylococci (0.42x10²±0.84 - 5.62x10²±1.07 CFU/ml) prevailed. The sanitary indicative species Escherichia coli was abundant, especially in the surface water layers (2.92x10²±5.54 CFU/ml). Pseudomonas species were less (0.30x10²±0.51 CFU/ml) and found only in the surface layer. Clostridia were isolated from 40% of surface water samples and 80% of bottom samples, but the sanitary indicative species C. perfringens was not detected. The isolated microorganisms were in greater quantities in the surface water layer, but the differences were statistically significant only for staphylococci. Fungi and clostridia were established in greater quantity at the bottom of the lake. [ABSTRACT FROM AUTHOR]

Vaginal diseases such as trichomoniasis, vulvovaginal candidiasis (VVC) and bacterial vaginosis (BV) are widespread worldwide. Accurate detection and administration of effective medications are essential, since such infections are related to negative effects for women during pregnancy and for newborns However, the presence of Escherichia coli in such infections is controversial, and hence it is important to identify these genetic and typical characteristics of E. coli strains isolated from genital infection, this review will focus on the diagnosis of E. coli isolates by 16SrRNA, discovering the effectiveness of some antimicrobials and studying some genes of virulence factors that affect the female reproductive system, In addition to its effect on fetuses in pregnant women. include with its impression share (fimH), type 1 fimbriae (%76.36), (cnf1), cytotoxic,necrotizing factor (36.36%), (usp) uropathogen specific protein (58.18%) and (ibeA), invation brain endothelium (32.72%), from (300) female genital tract secretions specimens isolate,The number of bacterial isolates was 165 (55%) and the number of yeast isolates was 110 (36,6%) no growth 25(8,33%) and number of E.coil isolates(55),The results also showed that the resistance and sensitivity of the different types of antimicrobial such as,amoxicillin-clavulanic acid(AMC) (54.54%) doxycycilin (DO) (32.72%),cefixime (CFM)(54.54%) ceftriaxone(CRO)(50.90%), cephalothin (KF) (14.54%),Cefotaxime (CTX) (54.54%),Nitrofurantoin (NI) (9.09%),ciprofloxacin (CIP) (78.18%), gentamicin (CN) (41.81%) trimethoprim /sulfamethoxazole (TS) (27.27%),imipenem(IPM) (89.09%) meropenem,(MEM) (90.90%), Amikacin (AK) (58.18%),Netilmicin (NET) (32.72%),lincomycin (L),(14.54%) Tobramycin (TN) (20%). [ABSTRACT FROM AUTHOR]

Abstract In this study real-time PCR assays were evaluated for the detection of enteroaggregative hemorrhagic Escherichia coli (EAHEC) O104:H4 in artificially contaminated mung bean and/alfalfa sprouts inoculated with 1, 10, and 100 CFU of EAHEC O104:H4 per 25 g sample (20, 10, and 2 replicates respectively). After selective culture enrichment the samples were tested using commercial real-time PCR kits detecting agg R/ aai C, stx / eae , and wzx O104. Using the commercial real-time PCR kits, the artificially contaminated samples were detected in the range of 75–80% positive results when contaminated with approximately 1 CFU, and 100% at 10 and 100 CFU. Microbiological detection employing O104-specific immunomagnetic capture and plating onto chromogenic media (modified Rainbow Agar and CHROMagar STEC) and confirmation by latex agglutination and PCR gave similar results (Cohen's kappa value between 0.61 and 1). In addition, the real-time PCR assay targeting the agg R and aai C genes, indicative of enteroaggregative Escherichia coli (EAggEC), was tested against a panel of 60 bacterial strains and demonstrated 100% exclusivity (54 strains) and 100% inclusivity (6 strains). This study demonstrates the efficacy of the real-time PCR assays for the specific and sensitive detection of EAHEC from spouts. Graphical abstract Unlabelled Image Highlights • Background bacterial population of sprouts grows during the storage phase at 4 °C. • The PCR assay was specific for detection of the O104 strains. • The detection limit for the PCR assays was 1 CFU/25 g in sprouts. • Microbiological and PCR-methods were comparable in terms of performance. [ABSTRACT FROM AUTHOR]

Abstract Immobilization of proteins on a biocompatible conductive interface is highly desirable for the fabrication of biosensors. In this study, a nanocomposite has been prepared by assembling well-distributed gold nanoparticles (AuNPs) on the surface of a polypyrrole-reduced graphene oxide (PPy-rGO) composite through electrostatic adsorption. This serves as a platform for immobilization of a capture antibody, which was conjugated onto the ferrocene doped polypyrrole-gold nanoparticles (PPy@Fc/AuNPs) composite. The design and performance of the biosensor was tested against detection of a whole-cell bacteria E. coli K12. This nanocomposite has a high surface area, good conductivity and biocompatibility, which is shown to be very suitable for enzyme-free detection of this bacteria. Results show excellent analytical performance with a linear range from 1.0 × 101 to 1.0 × 107 CFU mL–1 and a low detection limit of 10 CFU mL–1. The sensor has high selectivity, excellent reproducibility, and good stability. Graphical abstract fx1 Highlights • Gold nanoparticles were dispersed on the polypyrrole surface uniformly. • The high conductive and biocompatible PPy-rGO/AuNPs nanocomposite was used as an interface to immobilize antibodies. • Ferrocene doped polypyrrole-gold nanoparticles composite was used to generate current signal. • An enzyme-free sandwich immunosensor was constructed with good analytical performance. [ABSTRACT FROM AUTHOR]

An optical fiber based immunosensing platform formed by two identical chirped long period gratings (CLPGs) for sensitive and label-free detection of Escherichia coli (E. coli) is presented. The proposed immunosensor having two CLPGs with an inter-grating space (IGS) works like a Mach-Zehnder interferometer. The important feature of this sensor is that by changing the refractive index (RI) induced phase at the IGS region, a corresponding shift in resonance wavelength can be realized without affecting the actual grating region. Additionally, the confining bandwidth of the interference fringes promotes greater resolution in the resonance wavelength analysis of the transmission spectrum. The main aim of this study was the specific and rapid detection of the E. coli bacteria in phosphate buffer saline (detection range: 10 cfu/ml to 60 cfu/ml) by using the bio-functionalized IGS region as a sensing probe of the dual CLPG structure. The observed detection limit was 7 cfu/ml. For specificity analysis, Salmonella typhimurium and Staphylococcus aureus were tested and no significant shift in resonance wavelength was observed. In addition, the proposed immunosensor has the ability to selectively detect E. coli in real samples including lake water. The high specificity, good sensitivity to ambient RI, and robustness of the developed sensing platform will open a new avenue in optical immunosensing technology, and cascaded long CLPG sensors can contribute significantly to the detection of pathogenic bacteria in water and food samples. [ABSTRACT FROM AUTHOR]

We have previously generated four replicate populations of ionizing radiation (IR)-resistant Escherichia coli though directed evolution. Sequencing of isolates from these populations revealed that mutations affecting DNA repair (through DNA double-strand break repair and replication restart), ROS amelioration, and cell wall metabolism were prominent. Three mutations involved in DNA repair explained the IR resistance phenotype in one population, and similar DNA repair mutations were prominent in two others. The remaining population, IR-3-20, had no mutations in the key DNA repair proteins, suggesting that it had taken a different evolutionary path to IR resistance. Here, we present evidence that a variant of the anaerobic metabolism transcription factor FNR, unique to and isolated from population IR-3-20, plays a role in IR resistance. The F186I allele of FNR exhibits a diminished ability to activate transcription from FNR-activatable promoters, and furthermore reduces levels of intracellular ROS. The FNR F186I variant is apparently capable of enhancing resistance to IR under chronic irradiation conditions, but does not increase cell survival when exposed to acute irradiation. Our results underline the importance of dose rate on cell survival of IR exposure. [ABSTRACT FROM AUTHOR]

Background: Rapid and accurate identification of Verotoxigenic Escherichia coli (VTEC) O157:H7 is dependent on well-established, standardized and highly discriminatory typing methods. Currently, conventional subtyping tests for foodborne bacterial pathogen surveillance are rapidly being replaced with whole-genome sequencing (WGS) in public health laboratories. The capacity of WGS to revolutionize global foodborne disease surveillance has positioned this tool to become the new gold standard; however, to ensure evidence standards for public health decision making can still be achieved, the performance of WGS must be thoroughly validated against current gold standard methods prior to implementation. Here we aim to verify the performance of WGS in comparison to pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem repeat analysis (MLVA) for eight retrospective outbreaks of VTEC O157:H7 from the Canadian perspective. Since real-time implementation and routine use of WGS in public health laboratories is highly reliant on standardized data analysis tools, we also provide a comparative analysis of two popular methodologies for WGS analyses; an in-house developed single nucleotide variant phylogenomics (SNVPhyl) pipeline and the BioNumerics whole genome multilocus sequence typing (wgMLST) tool. To provide a useful and consistent starting point for examining laboratory-based surveillance data for VTEC O157:H7 in Canada, we also aim to describe the number of genetic differences observed among outbreak-associated isolates. Results: WGS provided enhanced resolution over traditional subtyping methods, and accurately distinguished outbreak-related isolates from non-outbreak related isolates with high epidemiological concordance. WGS also illuminated potential linkages between sporadic cases of illness and contaminated food, and isolates spanning multiple years. The topologies generated by SNVPhyl and wgMLST were highly congruent with strong statistical support. Few genetic differences were observed among outbreak-related isolates (≤5 SNVs/ < 10 wgMLST alleles) unless the outbreak was suspected to be multi-strain. Conclusions: This study validates the superiority of WGS and indicates the BioNumerics wgMLST schema is suitable for surveillance and cluster detection of VTEC O157:H7. These findings will provide a useful and consistent starting point for examining WGS data for prospective laboratory-based surveillance of VTEC O157:H7, but however, the data will continue to be interpreted according to context and in combination with epidemiological and food safety evidence to inform public-health decision making in Canada. [ABSTRACT FROM AUTHOR]

We have developed a label-free, enzyme-free, modification-free and DNA extraction-free fluorescent aptasensing (LEFA) method for detection of E. coli O157:H7 based on G-quadruplex formation using two ingeniously designed hairpin probes (GHP1 and GHP2). In the presence of E. coli O157:H7, it released the single stranded initiation sequence (IS) resulting in the toehold strand displacement between GHP1 and GHP2, which in turn led to the cyclic reuse of the production of DNA assemblies with numerous G-quadruplex structures and initiation sequences. Then these G-quadruplex structures can be recognized quickly by N -methyl mesoporphyrin IX (NMM) resulting in significantly enhanced fluorescence. The LEFA method was successfully implemented for detecting E. coli O157:H7 with a detection limit of 66 CFU/mL in pure culture, 10 CFU/mL and 1 CFU/mL after pre-incubation of the milk and tap water for 4 and 8 h, respectively. Moreover, the strategy could distinguish viable E. coli O157:H7 from dead E. coli O157:H7 and other species of pathogen cells. Furthermore, the whole process of the strategy is accomplished within 100 min. The results indicated that the approach may be used to effectively control potential microbial hazards in human health, food safety, and animal husbandry. [ABSTRACT FROM AUTHOR]

Herein, we report the development of an electrochemical biosensor for Escherichia coli O157:H7 diagnostic based on amino-functionalized metal–organic frameworks (MOFs) as a new generation of organic–inorganic hybrid nanocomposites. The electrical and morphological properties of MOFs were enhanced by interweaving each isolated MOF crystal with polyaniline (PANI). Subsequent attachment of the amine-modified aptamer to the polyanilinated MOFs was accomplished using glutaraldehyde (GA) as a cross-linking agent. The prepared biocompatible platform was carefully characterized by means of field-emission scanning electron microscopy (FESEM), energy-dispersive spectroscopy (EDS), Fourier transform infrared spectroscopy (FT-IR), and X-ray powder diffraction (XRD) techniques. The biosensor fabrication and its electrochemical characterizations were monitored by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques. Differential pulse voltammetry (DPV) was applied to monitoring and quantitation of the interaction between the aptamer and E. coli O157:H7 using methylene blue (MB) as an electrochemical indicator. Changes in the reduction peak current of MB in the presence of E. coli O157:H7 was recorded as an analytical signal and indicated a relationship with the logarithm of the E. coli O157:H7 concentration in the range of 2.1 × 101 to 2.1 × 107 CFU mL−1 with a LOQ of 21 CFU mL−1 and LOD of 2 CFU mL−1. The electrochemical aptasensor displayed good recovery values for the detection of E. coli O157:H7 in environmental real samples and also could act as a smart device to investigate the effects of antibacterial agents against E. coli O157:H7. [ABSTRACT FROM AUTHOR]

As strain variation and drug resistance become more pervasive, the prevention and control of infection have been a serious problem in recent years. The detection of pathogen is one of the most important parts of the process of diagnosis. Having a series of advantages, such as rapid response, high sensitivity, ease of use, and low cost, biosensors have received much attention and been studied deeply. Moreover, relying on its characteristics of small size, real time, and multiple analyses, biosensors have developed rapidly and used widely and are expected to be applied for microbiological detection in order to meet higher accuracy required by clinical diagnosis. The main goal of this contribution is not to simply collect and list all papers related to pathogen detection based on biosensors published recently, but to discuss critically the development and application of many kinds of biosensors such as electrochemical (amperometric, impedimetric, potentiometric, and conductometric), optical (fluorescent, fibre optic and surface plasmon resonance), and piezoelectric (quartz crystal microbalances and atomic force microscopy) biosensors in pathogen detection as well as the comparisons with the existing clinical detection methods (traditional culture, enzyme-linked immunosorbent assay, polymerase chain reaction, and mass spectrometry). [ABSTRACT FROM AUTHOR]

Whole cell MALDI is regularly used for the identification of bacteria to species level in clinical Microbiology laboratories. However, there remains a need to rapidly characterize and differentiate isolates below the species level to support outbreak management. We describe the implementation of a modified preparative approach for MALDI-MS combined with a custom analytical computational pipeline as a rapid procedure for subtyping Shigatoxigenic E. coli (STEC) and accurately identifying strain-specifying biomarkers. The technique was able to differentiate E. coli O157:H7 from other STEC. Within O157 serotype O157:H7 isolates were readily distinguishable from Sorbitol Fermenting O157 isolates. Overall, nine homogeneous groups of isolates were distinguished, each exhibiting distinct profiles of defining mass spectra features. This offers a robust analytical tool useable in reference/diagnostic public health scenarios. [ABSTRACT FROM AUTHOR]

The dynamic changes in free-chlorine levels within a commercial shredded lettuce washing process and implications for preventing cross-contamination by human pathogens during processing has been determined. The commercial lettuce processing line incorporated a pre-wash in Wash Tank 1 followed by a biocidal wash in Wash Tank 2. Within the commercial wash process, the free-chlorine concentration fluctuated in a cyclic pattern (ranging from 4 to 27 ppm) in Wash Tank 1 but was depleted within Wash Tank 2 (low as 0.1 ppm). Despite the variation in free-chlorine levels the Oxidation Reduction Potential of the wash tanks remained stable and consequently there was no significant correlation to free-chlorine levels and ORP (r 2 = 0.37). From analysis of the spent wash water it was found the Chemical Oxygen Demand (COD) was significantly higher in Wash Tank 1 (>5000 mg/l) compared to Wash Tank 2 (800–1001 mg/l). Significantly, both wash tanks operated below the chlorine demand that partly explained the inability to maintain constant free-chlorine levels. In laboratory studies, the spent wash water (containing disinfection byproducts) from the Wash Tanks supported inactivation of Salmonella (>3 log cfu reduction), Listeria monocytogenes (>4 log cfu reduction) and Escherichia coli O157:H7 (1–3 log cfu reduction). By adding chlorine beyond the chlorine breakpoint it was possible to achieve >4 log cfu reduction of the aforementioned pathogens, in addition to minimizing cross-contamination events between batches. The study has illustrated that commercial wash tanks for leafy greens operate below the chlorine breakpoint and hence maintaining specific free chlorine levels would be an unsuitable monitoring metric. Although disinfection byproducts contributed to antimicrobial capacity of the wash tank water it would be challenging to designate a specific limit of free-chlorine for purpose of process monitoring. [ABSTRACT FROM AUTHOR]

Colloidal gold immunochromatographic assay (ICA) has poor sensitivity when used for Escherichia coli O157:H7 ( E. coli O157:H7) detection. Eu (III)-doped polystyrene nanoparticle (EuNP) has a large range of stokes shift, long decay time, and wide excitation spectrum and narrow emission spectra. EuNP has been used as novel probe in ICA to improve sensitivity. In this study, carboxyl-modified EuNPs were prepared with different linkers. ICA based on EuNP, EuNP-6 carbon chain (CC) complex, EuNP-200CC complex, EuNP-1000CC complex, and EuNP-streptavidin (EuNP-SA) complex were systematically compared for the detection of E. coli O157:H7. Under optimized working conditions, the limits of detection (LOD) of EuNP-ICA, EuNP-6CC-ICA, EuNP-200CC-ICA, EuNP-1000CC-ICA, and EuNP-SA-ICA were 9.54 × 10 2 , 1.59 × 10 2 , 3.18 × 10 2 , 2.98 × 10 2 , and 1.08 × 10 2 colony-forming units (CFU) mL −1 , respectively. The linear ranges of EuNP-ICA, EuNP-6CC-ICA, EuNP-200CC-ICA, EuNP-1000CC-ICA, and EuNP-SA-ICA were 6.36 × 10 2 –1.59 × 10 5 , 3.18 × 10 2 –1.59 × 10 5 , 6.36 × 10 2 –1.59 × 10 5 , 6.36 × 10 2 –1.59 × 10 5 , and 8.0 × 10 1 –1.59 × 10 5 CFU mL −1 , respectively. EuNP-SA-ICA exhibited the highest sensitivity and the widest linear range with good specificity, accuracy, and precision. It could be a promising analytical method for detecting E. coli O157:H7 in food samples. EuNP-SA-ICA may be a good model for detecting low concentrations of other food-borne pathogens. [ABSTRACT FROM AUTHOR]

Dairy cow effluent collected over winter from a loose-housed barn was applied to a series of large infield plots (400 m2) using low rate and low depth (LRLD) application methods. Applications were confined to the winter period, at a time when soil moisture content was often at or very near to field capacity and was applied over two seasons. Cows were confined to the housing facility during winter only, and outside of this period they remained on pasture. Losses of nitrogen (N), phosphorus (P) and the faecal indicator bacteriumEscherichia coli(E. coli) in surface runoff and subsurface drainage from the LRLD treatment were compared with losses from effluent applications that occurred during spring to autumn, at an application depth not exceeding the soil water deficit, i.e. a standard practice treatment (SP, typically 10–15 mm per application). The annual quantities of nutrients applied by the treatments and the grazing managements imposed were similar. Although winter losses of N were significantly greater for the LRLD treatment (15 vs. 8 kg N ha−1for the SP treatment), on an annual basis fluxes were similar between treatments (approximately 20 kg N ha−1year−1). Effluent management had no significant effect on the annual fluxes of P andE. colialthough the latter varied considerably. Average contaminant fluxes over a 2-year period indicated that the LRLD management system did not lead to a significantly greater risk to water quality compared with standard practices. [ABSTRACT FROM PUBLISHER]

Capillary zone electrophoresis-electrospray ionizationtandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as an invaluable platform for top-down proteomics. However, the scale of top-down proteomics using CZE-MS/MS is still limited due to the low loading capacity and narrow separation window of CZE. In this work, for the first time we systematically evaluated the dynamic pH junction method for focusing of intact proteins during CZE-MS. The optimized dynamic pH junction-based CZE-MS/MS approached a 1 μL loading capacity, 90 min separation window, and high peak capacity (~280) for characterization of an Escherichia coli proteome. The results represent the largest loading capacity and the highest peak capacity of CZE for topdown characterization of complex proteomes. Single-shot CZE-MS/MS identified about 2800 proteoform-spectrum matches, nearly 600 proteoforms, and 200 proteins from the Escherichia coli proteome with spectrum-level false discovery rate (FDR) less than 1%. The number of identified proteoforms in this work is over three times higher than that in previous single-shot CZE-MS/MS studies. Truncations, N-terminal methionine excision, signal peptide removal, and some post-translational modifications including oxidation and acetylation were detected. [ABSTRACT FROM AUTHOR]

Escherichia coli (E. coli) O157:H7 is a foodborne pathogen that causes symptoms in humans. Its rapid identification should be considered to avoid toxic effects of the pathogen. In this study, systematic evolution of ligands by exponential enrichment using whole cells (Cell-SELEX) method was used for recognizing E. coli strain, O157 by single-stranded DNA library of aptamer. Nine rounds of cell-selex procedure were applied using O157, as a whole-cell target, with O42, K12, Top10, DH5α E. coli cells, Shigella flexneri and Salmonella typhi as counterparts. The specific interaction between selected DNA aptamers and targeted cell was assessed. After applying six rounds of SELEX for selection of DNA aptamers, the candidate sequences were obtained. Finally, specific aptamer was selected as an ideal aptamer for detection and capturing of E. coli O157. Dissociation constant of the selected aptamer were calculated (107.6 ± 67.8 pM). In addition, the secondary structure prediction and cross reactivity assays were performed. The isolated aptamer efficiency was confirmed and it was shown that the new DNA aptamer sequence has the ability to use for detection. This specific O157:H7 aptamer have the potential for application as a diagnostic ligand and could be used for detection of the related food borne diseases. [ABSTRACT FROM AUTHOR]

Transcriptional regulatory networks (TRNs) have been studied intensely for >25 y. Yet, even for the Escherichia coli TRN--probably the best characterized TRN--several questions remain. Here, we address three questions: (i) How complete is our knowledge of the E. coli TRN; (ii) how well can we predict gene expression using this TRN; and (iii) how robust is our understanding of the TRN? First, we reconstructed a high-confidence TRN (hiTRN) consisting of 147 transcription factors (TFs) regulating 1,538 transcription units (TUs) encoding 1,764 genes. The 3,797 high-confidence regulatory interactions were collected from published, validated chromatin immunoprecipitation (ChIP) data and RegulonDB. For 21 different TF knockouts, up to 63% of the differentially expressed genes in the hiTRN were traced to the knocked-out TF through regulatory cascades. Second, we trained supervised machine learning algorithms to predict the expression of 1,364 TUs given TF activities using 441 samples. The algorithms accurately predicted condition-specific expression for 86% (1,174 of 1,364) of the TUs, while 193 TUs (14%) were predicted better than random TRNs. Third, we identified 10 regulatory modules whose definitions were robust against changes to the TRN or expression compendium. Using surrogate variable analysis, we also identified three unmodeled factors that systematically influenced gene expression. Our computational workflow comprehensively characterizes the predictive capabilities and systems-level functions of an organism's TRN from disparate data types. [ABSTRACT FROM AUTHOR]

For the first time, the antibacterial activity of ethylene glycol (EG), a routine frequently used solvent against Escherichia coli ( E. coli ) bacterium, was assessed. The antibacterial activity of EG against E. coli was measured using colony counting and broth turbidity assays. The influence of EG concentration (1.5–25.0%v/v) and exposure time on the growth of E. coli was investigated. By increasing EG concentration, its antibacterial activity against E. coli increased so that for 24.0% of EG, the bacteria growth was totally inhibited within 4 h. The MIC and MBC values of EG are 18.0 and 24.0%v/v, respectively. Since the ratio of MBC to MIC is less than four, EG acts as a bactericidal agent. Also, a model for the slopes of the linear part of the growth curves was proposed. The SEM images of bacteria cells before and after exposure to EG show that most E. coli were seriously distorted. [ABSTRACT FROM AUTHOR]

Background: Diagnosis of enterotoxigenic E. coli (ETEC) associated diarrhea is complicated by the diversity of E.coli virulence factors. This study developed a multiplex quantitative PCR assay based on high-resolution melting curves analysis (HRMqPCR) to identify and quantify genes encoding five ETEC fimbriae related to diarrhea in swine, i.e. K99, F41, F18, F6 and K88. Methods: Five fimbriae expressed by ETEC were amplified in multiple HRM-qPCR reactions to allow simultaneous identification and quantification of five target genes. The assay was calibrated to allow quantification of the most abundant target gene, and validated by analysis of 30 samples obtained from piglets with diarrhea and healthy controls, and comparison to standard qPCR detection. Results: The five amplicons with melting temperatures (Tm) ranging from 74.7 ± 0.06 to 80.5 ± 0.15 °C were wellseparated by HRM-qPCR. The area of amplicons under the melting peak correlated linearly to the proportion of the template in the calibration mixture if the proportion exceeded 4.8% (K88) or <1% (all other amplicons). The suitability of the method was evaluated using 30 samples fromweaned pigs aged 6–7 weeks; 14 of these animals suffered from diarrhea in consequence of poor sanitary conditions. Genes encoding fimbriae and enterotoxins were quantified by HRMqPCR and/or qPCR. The multiplex HRM-qPCR allowed accurate analysis when the total gene copy number of targets was more than 1 × 105 / g wet feces and the HRM curves were able to simultaneously distinguish fimbriae genes in the fecal samples. The relative quantification of the most abundant F18 based on melting peak area was highly correlated (P < 0.001; r² = 0.956) with that of individual qPCR result but the correlation for less abundant fimbriae was much lower. Conclusions: The multiplex HRM assay identifies ETEC virulence factors specifically and efficiently. It correctly indicated the predominant fimbriae type and additionally provides information of presence/ absence of other fimbriae types and it could find broad applications for pathogen diagnosis. [ABSTRACT FROM AUTHOR]

Background: Acute diarrhea is one of the major public health problems worldwide. Most of studies on acute diarrhea have been made on infants aged below 5 years and few efforts have been made to identify the etiological agents of acute diarrhea in people over five, especially in China. Methods: 271 diarrhea cases and 149 healthy controls over 5 years were recruited from four participating hospitals between June 2014 and July 2015. Each stool specimen was collected to detect a series of enteric pathogens, involving five viruses (Rotavirus group A, RVA; Norovirus, NoV; Sapovirus, SaV; Astrovirus, As; and Adenovirus, Ad), seven bacteria (diarrheagenic Escherichia coli, DEC; non-typhoidal Salmonella, NTS; Shigella spp.; Vibrio cholera; Vibrio parahaemolyticus; Aeromonas spp.; and Plesiomonas spp.) and three protozoa (Cryptosporidium spp., Giardia lamblia, G. lamblia, and Blastocystis hominis, B. hominis). Standard microbiological and molecular methods were applied to detect these pathogens. Data was analyzed using Chi square, Fisher-exact tests and logistic regressions. Results: The prevalence of at least one enteric pathogen was detected in 29.2% (79/271) acute diarrhea cases and in 12.1% (18/149) in healthy controls (p < 0.0001). Enteric viral infections (14.4%) were the most common in patients suffering from acute diarrhea, followed by bacteria (13.7%) and intestinal protozoa (4.8%). DEC (12.5%) was the most common causative agent in diarrhea cases, followed by NoV GII (10.0%), RVA (7.4%) and B. hominis (4.8%). The prevalence of co-infection was statistically higher (p = 0.0059) in the case group (7.7%) than in the healthy control (1.3%). RVA-NoV GII (3.0%) was the most common co-infection in symptomatic cases. Conclusions: DEC was the most predominant pathogen in diarrhea cases, but it was largely overlooked because the lack of laboratory capacities. Because of the high prevalence of co-infections, it is recommended the urgent development of alternative laboratory methods to assess polymicrobial infections. Such methodological improvements will result in a better prevention and treatment strategies to control diarrhea illness in China. [ABSTRACT FROM AUTHOR]

The prevalence and behavior of multidrug-resistant diarrheagenic Escherichia coli pathotypes on coriander was determined. One hundred coriander samples were collected from markets. Generic E. coli were determined using the most probable number procedure. Diarrheagenic E. coli pathotypes (DEPs) were identified using two multiplex polymerase chain reaction procedures. Susceptibility to sixteen antibiotics was tested for the isolated DEPs strains by standard test. The behavior of multidrug-resistant DEPs isolated from coriander was determined on coriander leaves and chopped coriander at 25°± 2 °C and 3°± 2 °C. Generic E. coli and DEPs were identified, respectively, in 43 and 7% of samples. Nine DEPs strains were isolated from positive coriander samples. The identified DEPs included Shiga toxin-producing E. coli (STEC, 4%) enterotoxigenic E. coli (ETEC, 2%) and enteropathogenic E. coli (EPEC, 1%). All isolated DEPs strains exhibited multi-resistance to antibiotics. On inoculated coriander leaves stored at 25°± 2 °C or 3°± 2 °C, no growth was observed for multidrug-resistant DEPs strains. However, multidrug-resistant DEPs strains grew in chopped coriander: after 24 h at 25° ± 2 °C, DEPs strains had grown to approximately 3 log CFU/g. However, at 3°± 2 °C the bacterial growth was inhibited. To the best of our knowledge, this is the first report of the presence and behavior of multidrug-resistant STEC, ETEC and EPEC on coriander and chopped coriander. [ABSTRACT FROM AUTHOR]

Bacteriophage (phage) amplification is an attractive method for the detection of bacteria due to a narrow phage-host specificity, short amplification times, and the phages’ ability to differentiate between viable and non-viable bacterial cells. The next step in phage-based bacteria detection is leveraging bioengineered phages to create low-cost, rapid, and easy-to-use detection platforms such as lateral flow assays. Our work establishes the proof-of-concept for the use of bioengineered T7 phage strains to increase the sensitivity of phage amplification-based lateral flow assays. We have demonstrated a greater than 10-fold increase in sensitivity using a phage-based protein reporter, maltose-binding protein, over the detection of replicated T7 phage viron itself, and a greater then 100-fold increase in sensitivity using a phage-based enzymatic reporter, alkaline phosphatase. This increase in sensitivity enabled us to detect 10 3 CFU/mL of Escherichia coli in broth after 7 h, and by adding a filter concentration step, the ability to detect a regulatory relevant E. coli concentration of 100 CFU/100 mL in inoculated river water after 9 h, where the current standard requires days for results. The combination of the paper fluidic format with phage-based detection provides a platform for the development of novel diagnostics that are sensitive, rapid, and easy to use. [ABSTRACT FROM AUTHOR]

This study was designed to determine the shiga toxin producing genes and to investigate antibiotic sensitivity or resistant patterns of the Escherichia coli isolated from diarrheic children at Mymensingh Medical College Hospital, Bangladesh. A total of 83 stool samples were collected and screened for the detection of E. coli on the basis of cultural, staining and biochemical properties followed by molecular detection by Polymerase Chain Reaction (PCR) using genus specific 16SrRNA primers. Antimicrobial susceptibility pattern of E. coli was determined by disc diffusion method against 9 antimicrobial agents. In this study, 27 (32.53%) out of 83 samples, were confirmed as E. coli. Overall prevalence of shiga toxin producing E. coli (STEC) among the examined children was 1.20% (n=1/83). Further, 27 E. coli isolates were analyzed for the presence of Stx-1 and Stx-2 genes by duplex-PCR. The STEC isolate was confirmed to be positive for the presence of the Stx-2 gene only. Highest susceptibility of the E. coli isolates was found against Gentamicin (92.59%), followed by Ciprofloxacin (48.14%) and Moxifloxacin (33.33%). More than 77.78% of the isolates were resistant to more than three antibiotics thus defined as multidrug resistant (MDR). In conclusion, Gentamicin and Ciprofloxacin can be recommended as the effective drugs successful treatment of STEC infections in children. [ABSTRACT FROM AUTHOR]

The study was intended for molecular detection of E. coli isolated from raw cow's milk. A total of 20 milk samples were collected from different upazila markets of Jamalpur, Tangail, Kishoreganj and Netrokona districts of Bangladesh. Milk samples were cultured onto various culture media for the isolation of bacteria. The isolated bacteria were identified by studying staining characteristics, cultural properties on different selective media, biochemical tests, catalase and coagulase test, and finally by PCR. Out of 20 samples, 15 (75%) milk samples were found positive for E. coli. 15 Escherichia coli isolates were amplified by 16S rRNA gene based PCR. Antimicrobial sensitivity test was carried out to ascertain the susceptibility of the organism to various antibiotics. Its results showed that the E. coli isolates were resistant to amoxycillin (86.67%) and erythromycin (73.33%) but sensitive to azithromycin (53.33%), ciprofloxacin (86.67%), gentamicin (86.67%), norfloxacin (80%) and streptomycin (66.67%). [ABSTRACT FROM AUTHOR]

The study was conducted to isolate and identify Escherichia coli naturally occurring in healthy chickens. The study was also focused on the investigation of drug sensitivity and resistance pattern of the isolates. A total number of 50 field samples (cloacal swab) were collected from apparently healthy native chickens from different areas of the Sylhet region. 10 samples from Bandarbazar, 10 samples from Ambarkhana bazar, 10 samples from Eidgah bazar, 10 samples from Baluchor bazar and 10 samples from University residential area. Out of these 50 samples of apparently healthy chicken the number of samples found to be positive for Escherichia coli were 4 of Bandar bazaar samples , 4 of Ambarkhana bazaar samples , 3 of Eidgah bazaar samples , 3 of University residential area samples and 2 of Baluchor bazaar samples . This study reveals that the prevalence rate of E.coli is 40% of Bandar bazaar , 40% of Ambarkhana bazaar ,30% of Eidgah bazaar , 30% of University residential area and 20% of Baluchor bazaar . All of the Escherichia coli isolates revealed the same morphological, cultural and biochemical characteristics. The antibiotic sensitivity pattern showed that among the Escherichia coli isolates (16 samples) highly sensitive to ceftriaxone, intermediate sensitive to nalidixic acid, cephalexin, gentamycin, erythromycin and chloramphenicle and fully resistant to penicillin and bacitracin. It may be concluded that the high resistance of Escherichia coli to antibiotic may constitutes a potential threat to chicken health and consumption of public. [ABSTRACT FROM AUTHOR]

Most Escherichia coli strains are nonpathogenic. However, for clinical diagnosis and food safety analysis, current identification methods for pathogenic E. coli either are time-consuming and/or provide limited information. Here, we utilized a custom DNA microarray with informative genetic features extracted from 368 sequence sets for rapid and high-throughput pathogen identification. The FDA Escherichia coli Identification (FDA-ECID) platform contains three sets of molecularly informative features that together stratify strain identification and relatedness. First, 53 known flagellin alleles, 103 alleles of wzx and wzy, and 5 alleles of wzm provide molecular serotyping utility. Second, 41,932 probe sets representing the pan-genome of E. coli provide strain-level gene content information. Third, approximately 125,000 single nucleotide polymorphisms (SNPs) of available whole-genome sequences (WGS) were distilled to 9,984 SNPs capable of recapitulating the E. coli phylogeny. We analyzed 103 diverse E. coli strains with available WGS data, including those associated with past foodborne illnesses, to determine robustness and accuracy. The array was able to accurately identify the molecular O and H serotypes, potentially correcting serological failures and providing better resolution for H-nontypeable/nonmotile phenotypes. In addition, molecular risk assessment was possible with key virulence marker identifications. Epidemiologically, each strain had a unique comparative genomic fingerprint that was extended to an additional 507 food and clinical isolates. Finally, a 99.7% phylogenetic concordance was established between microarray analysis and WGS using SNP-level data for advanced genome typing. Our study demonstrates FDA-ECID as a powerful tool for epidemiology and molecular risk assessment with the capacity to profile the global landscape and diversity of E. coli. [ABSTRACT FROM AUTHOR]

Slightly acidic electrolyzed water (SAEW) has been recognized as an effective bactericidal agent with free chlorine, but its limitations include its instability and its great dependence on equipment. Newly developed circulating electrolyzed water (CEW) with a higher available chlorine concentration (ACC) could successfully overcome these limitations. In this study, SAEW (ACC of 20 mg/L), CEW1 (ACC of 200 mg/L), and CEW2 (ACC of 20 mg/L) were evaluated for changes in properties (pH, oxidization reduction potential [ORP], and ACC) during storage in open or closed glass bottles under light or dark conditions at room temperature (approximately 20 °C) and after washing pork and lettuce. Additionally, the washed pork and lettuce were evaluated for total viable counts, pH and general appearance. The results showed that CEW1 with a higher ACC has better stability than SAEW with a lower ACC for the storage and washing experiments, and CEW still remained stable after dilution with distilled water. The property indices of EW were greatly affected for the pork-washing experiments compared with the lettuce-washing experiments, probably due to the existence of alkaline and organic materials on the surface of pork. Furthermore, EWs were more effective for inactivating microbes in lettuce than in pork, while there was no significant difference in tissue pH and the general appearance of pork and lettuce. These findings indicated that CEW with a higher ACC shows potential for reducing foodborne pathogens on pork and lettuce without effects on their physicochemical characteristics, and it can be applied in a diluted form. [ABSTRACT FROM AUTHOR]

Standard methods to detect Escherichia coli contamination in food use the polymerase chain reaction (PCR) and agar culture plates. These methods require multiple incubation steps and take a long time to results. An improved rapid flow-cytometry based detection method was developed, using a fluorescence-labeled oligonucleotide probe specifically binding a16S rRNA sequence. The method positively detected 51 E. coli isolates as well as 4 Shigella species. All 27 non-E. coli strains tested gave negative results. Comparison of the new genetic assay with a total plate count (TPC) assay and agar plate counting indicated similar sensitivity, agreement between cytometry cell and colony counts. This method can detect a small number of E.coli cells in the presence of large numbers of other bacteria. This method can be used for rapid, economical, and stable detection of E. coli and Shigella contamination in the food industry and other contexts. [ABSTRACT FROM AUTHOR]

Objectives: The objective of this study was to determine the population structure of Escherichia coli ST73 isolated from human bacteraemia and urinary tract infections. Methods: The genomes of 22 E. coli ST73 isolates were sequenced using the Illumina HiSeq platform. Highresolution SNP typing was used to create a phylogenetic tree. Comparative genomics were also performed using a pangenome approach. In silico and S1-PFGE plasmid profiling was conducted, and isolates were checked for their ability to survive exposure to human serum. Results: E. coli ST73 isolates circulating in clinically unrelated episodes show a high degree of diversity at a whole-genome level, but exhibit conservation in gene content, particularly in virulence-associated gene carriage. The isolates also contain a highly diverse plasmid pool that confers MDR via carriage of CTX-M genes. Conclusions: Our data show that a rise in incidence of MDR E. coli ST73 clinical isolates is not due to a circulating outbreak strain as in E. coli ST131. Rather the ST73 circulating strains are distantly related and carry a diverse set of resistance plasmids. This suggests that the evolutionary events behind emergence of drug-resistant E. coli differ between lineages. [ABSTRACT FROM AUTHOR]

There remains a need for rapid, specific and sensitive assays for the detection of bacterial indicators for water quality monitoring. In this study, a strategy for rapid detection of Escherichia coli in drinking water has been developed. This strategy is based on the use of the substrate 4-methylumbelliferyl-β- d -glucuronide (MUG), which is hydrolyzed rapidly by the action of E. coli β- d -glucuronidase (GUD) enzyme to yield a fluorogenic 4-methylumbelliferone (4-MU) product that can be quantified and related to the number of E. coli cells present in water samples. In this study, the detection time required for the biosensor response ranged between 20 and 120 min, depending on the number of bacteria in the sample. This approach does not need extensive sample processing with a rapid detection capability. The specificity of the MUG substrate was examined in both, pure cultures of non-target bacterial genera such as Klebsiella , Salmonella , Enterobacter and Bacillus . Non-target substrates that included 4-methylumbelliferyl-β- d -galactopyranoside (MUGal) and l -leucine β-naphthylamide aminopeptidase (LLβ-N) were also investigated to identify nonspecific patterns of enzymatic activities in E. coli . GUD activity was found to be specific for E. coli and no further enzymatic activity was detected by other species. In addition, fluorescence assays were performed for the detection of E. coli to generate standard curves; and the sensitivity of the GUD enzymatic response was measured and repeatedly determined to be less than 10 E. coli cells in a reaction vial. The applicability of the method was tested by performing multiple fluorescence assays under pure and mixed bacterial flora in environmental samples. The results of this study showed that the fluorescence signals generated in samples using specific substrate molecules can be utilized to develop a bio-sensing platform for the detection of E. coli in drinking water. Furthermore, this system can be applied independently or in conjunction with other methods as a part of an array of biochemical assays in order to reliably detect E. coli in water. [ABSTRACT FROM AUTHOR]

We describe the use of lysozyme as a sensitive reporter for detection of bacteria. The assay comprises the following steps: First, specific primers are used for targeting the rDNA of E. coli and S. aureus, respectively, and the polymerase chain reaction is applied to amplify bacterial DNA. Then, the target DNA binds to the capture probe immobilized on magnetic beads, with lysozyme acting as signal reporter that catalyzes the hydrolysis of an enzyme substrate to form the blue fluorescent product 4-methylumbelliferone. Coupled with multiple lysozyme substrates, this fluorometric detecting method has a detection limit of 50 CFU⋅mL bacteria. The optical changes may also be detected by colorimetry and monitored with bare eyes. The method was selective over other bacteria and was successfully applied to the determination of E. coli and S. aureus in human serum, which provides a perspective for lysozyme application and species-specific pathogenic bacteria detection. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The SLAMF family (SLAMF) of cell surface glycoproteins is comprised of nine glycoproteins and while SLAMF1, 3, 5, 6, 7, 8, and 9 are self-ligand receptors, SLAMF2 and SLAMF4 interact with each other. Their interactions induce signal transduction networks in trans, thereby shaping immune cell--cell communications. Collectively, these receptors modulate a wide range of functions, such as myeloid cell and lymphocyte development, and T and B cell responses to microbes and parasites. In addition, several SLAMF receptors serve as microbial sensors, which either positively or negatively modulate the function of macrophages, dendritic cells, neutrophils, and NK cells in response to microbial challenges. The SLAMF receptor--microbe interactions contribute both to intracellular microbicidal activity as well as to migration of phagocytes to the site of inflammation. In this review, we describe the current knowledge on how the SLAMF receptors and their specific adapters SLAM-associated protein and EAT-2 regulate innate and adaptive immune responses to microbes. [ABSTRACT FROM AUTHOR]

Culture-independent diagnostics reduce the reliance on traditional (and slower) culture-based methodologies. Here we capitalize on advances in next-generation sequencing (NGS) to apply this approach to food pathogen detection utilizing NGS as an analytical tool. In this study, spiking spinach with Shiga toxin-producing Escherichia coli (STEC) following an established FDA culture- based protocol was used in conjunction with shotgun metagenomic sequencing to determine the limits of detection, sensitivity, and specificity levels and to obtain information on the microbiology of the protocol. We show that an expected level of contamination (-10 CFU/100 g) could be adequately detected (including key virulence determinants and strain-level specificity) within 8 h of enrichment at a sequencing depth of 10,000,000 reads. We also rationalize the relative benefit of static versus shaking culture conditions and the addition of selected antimicrobial agents, thereby validating the long-standing culture-based parameters behind such protocols. Moreover, the shotgun metagenomic approach was informative regarding the dynamics of microbial communities during the enrichment process, including initial surveys of the microbial loads associated with bagged spinach; the microbes found included key genera such as Pseudomonas, Pantoea, and Exiguobacterium. Collectively, our metagenomic study highlights and considers various parameters required for transitioning to such sequencing-based diagnostics for food safety and the potential to develop better enrichment processes in a high-throughput manner not previously possible. Future studies will investigate new species-specific DNA signature target regimens, rational design of medium components in concert with judicious use of additives, such as antibiotics, and alterations in the sample processing protocol to enhance detection. [ABSTRACT FROM AUTHOR]

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Escherichia coli O157 and O26 shedding patterns in cattle are known to vary widely. To address gaps in the understanding of the underlying factors which impact on shedding dynamics, sensitive and rapid quantitative methods which can be applied in surveillance studies on cattle are required. Current approaches for enumeration of verocytotoxigenic E. coli (VTEC) in cattle faeces are based on direct plating onto selective agars, most probable number (MPN) or real time PCR applied directly to faecal samples, all of which have limitations in terms of the labour involved or their sensitivity. The objective of this study was to develop a sensitive real time quantitative PCR assay, to quantify O157 and O26 in bovine recto-anal junction (RAJ) swabs. The approach was to target serogroup specific genes rfbE and wzx , and to couple a short enrichment, with the use of a standard calibration curve relating real time PCR cycle threshold (C t ) values against the initial concentration of the pathogen in the sample. Following initial experiments in broth culture, a 5 h enrichment in modified tryptone soya broth with novobiocin (20 mg/l) (mTSBn) was found to be optimal, and a linear correlation between inocula (Log 10 1 to 6 CFU ml − 1 ) and the PCR C t values for both E. coli O157 (R 2 = 0.99, rsd = 0.58) and E. coli O26 (R 2 = 0.99, rsd = 0.44) was confirmed. The developed method was then applied to bovine RAJ swab samples (n = 153), which were inoculated with E. coli O157 or O26 (Log 10 1 to 7 CFU swab − 1 ). Calibration curves yielded correlations for E. coli O157 of R 2 = 0.86, rsd = 0.72 and for O26 (R 2 = 0.88, rsd = 0.69). In conclusion, a sensitive method for detection and enumeration of two significant VTEC serogroups in bovine RAJ samples has been developed and validated, and will support studies on the bovine shedding dynamics of these pathogens in cattle. [ABSTRACT FROM AUTHOR]

Enterohemorrhagic Escherichia coli (EHEC) O121:H19 belong to a specific clonal type distinct from other classical EHEC and major enteropathogenic E. coli groups and is regarded as one of the major EHEC serogroups involved in severe infections in humans. Sequencing of the fliC genes associated with the flagellar antigen H19 (fliCH19) revealed the genetic diversity of the fliCH19 gene sequences in E. coli. A cluster analysis of 12 fliCH19 sequences, 4 from O121 and 8 from non-O121 E. coli strains, revealed five different genotypes. All O121:H19 strains fell into one cluster, whereas a second cluster was formed by five non-O121:H19 strains. Cluster 1 and cluster 2 strains differ by 27 single nucleotide exchanges in their fliCH19 genes (98.5% homology). Based on allele discrimination of the fliCH19 genes, a real-time PCR test was designed for specific identification of EHEC O121:H19. The O121 fliCH19 PCR tested negative in 73 E. coli H19 strains that belonged to serogroups other than O121, including 28 different O groups, O-nontypeable H19, and O-rough:H19 strains. The O121 fliCH19 PCR reacted with all 16 tested O121:H19 strains and 1 O-rough:H19 strain which was positive for the O121 wzx gene. A cross-reaction was observed only with E. coli H32 strains which share sequence similarities in the target region of the O121 fliCH19 PCR. The combined use of O-antigen genotyping (O121 wzx) and the detection of O121 fliCH19 allele type contributes to improving the identification and molecular serotyping of EHEC O121:H19 motile and nonmotile strains and variants of these strains lacking stx genes. [ABSTRACT FROM AUTHOR]

Background: The presence of coliform bacteria is routinely assessed to establish the microbiological safety of water supplies and raw or processed foods. Coliforms are a group of lactose-fermenting Enterobacteriaceae, which most likely acquired the lacZ gene by horizontal transfer and therefore constitute a polyphyletic group. Among this group of bacteria is Escherichia coli, the pathogen that is most frequently associated with foodborne disease outbreaks and is often identified by β-glucuronidase enzymatic activity or by the redundant detection of uidA by PCR. Because a significant fraction of essential E. coli genes are preserved throughout the bacterial kingdom, alternative oligonucleotide primers for specific E. coli detection are not easily identified. Results: In this manuscript, two strategies were used to design oligonucleotide primers with differing levels of specificity for the simultaneous detection of total coliforms and E. coli by multiplex PCR. A consensus sequence of lacZ and the orphan gene yaiO were chosen as targets for amplification, yielding 234 bp and 115 bp PCR products, respectively. Conclusions: The assay designed in this work demonstrated superior detection ability when tested with lab collection and dairy isolated lactose-fermenting strains. While lacZ amplicons were found in a wide range of coliforms, yaiO amplification was highly specific for E. coli. Additionally, yaiO detection is non-redundant with enzymatic methods. [ABSTRACT FROM AUTHOR]

A nanobiosensor chip, utilizing surface enhanced Raman spectroscopy (SERS) on nanosculptured thin films (nSTFs) of silver, was shown to detect Escherichia coli (E. coli) bacteria down to the concentration level of a single bacterium. The sensor utilizes highly enhanced plasmonic nSTFs of silver on a silicon platform for the enhancement of Raman bands as checked with adsorbed 4-aminothiophenol molecules. T-4 bacteriophages were immobilized on the aforementioned surface of the chip for the specific capture of target E. coli bacteria. To demonstrate that no significant non-specific immobilization of other bacteria occurs, three different, additional bacterial strains, Chromobacterium violaceum, Paracoccus denitrificans and Pseudomonas aeruginosa were used. Furthermore, experiments performed on an additional strain of E. coli to address the specificity and reusability of the sensor showed that the sensor operates for different strains of E. coli and is reusable. Time resolved phase contrast microscopy of the E. coli-T4 bacteriophage chip was performed to study its interaction with bacteria over time. Results showed that the present sensor performs a fast, accurate and stable detection of E. coli with ultra-small concentrations of bacteria down to the level of a single bacterium in 10 μl volume of the sample. [ABSTRACT FROM AUTHOR]

Bombyx mori , a lepidopteran insect, is one of the earliest models for pattern recognition of Gram-negative bacteria, which may induce the IMD pathway for production of antibacterial peptides. So far, several recognition proteins have been reported in B. mori . However, the connection between pattern recognition of Gram negative bacteria and activation of BmRelish1, a transcription factor controlled by the IMD pathway remains largely unknown. In the present study, we identify BmCPT1, a cuticle protein bearing a Tweedle domain. Its gene expression is co-regulated by NF-kappaB and juvenile hormone signals. BmCPT1 is induced by Escherichia coli in fat bodies and hemocytes, but is constitutively expressed in the epidermis. In vitro binding assays indicate that BmCPT1 protein recognizes and binds to E. coli peptidoglycan. Post-transcriptionally modified BmCPT1 in the hemolymph binds to E. coli cells through interactions with peptidoglycan recognition protein-5 (BmPGRP5) and lipopolysaccharide binding protein (BmLBP). Transgenic overexpression of BmCPT1 causes the upregulated expression of BmRelish1 and clear induction of two gloverin genes. Therefore, BmCPT1 may work along with BmPGRP-S5 and BmLBP to recognize E. coli in the hemolymph and indirectly activate BmRelish1 to induce antimicrobial peptide synthesis. [ABSTRACT FROM AUTHOR]

The removal of humic acid from soil samples is of great importance for the nucleic acid based detection of pathogenic bacteria because it is one of the main enzyme (polymerase) reaction inhibitors. A simple and effective detection tool that detects pathogens in fertilized soil samples was developed here by the combination of sample preparation steps (the concentration of bacteria and removal of humic acid) and micro amplification steps in a microfluidic pillar chip with a single chamber. The pH dependent ternary interactions between E. coli O157:H7 , humic acid, and solid surface were investigated and optimized for maximum adsorption of E. coli O157:H7 and desorption of humic acid on a solid surface to perform serial processes ((1) concentration of bacteria, (2) removal of humic acid, (3) nucleic acid extraction/amplification, and (4) fluorescent detection) in a microfluidic chip. These serial processes were successfully performed in a microfluidic PDMS chip with 400 micro pillars, resulting in the 87.5% rate of success in detection of 10 3 cfu of E. coli O157:H7 in fertilized soil (15 g) containing humic acid (up to 5000 ppm) for 2 h. [ABSTRACT FROM AUTHOR]

In this report, a new polythiophene interface is fabricated containing fused quinone moieties which are then glycosylated to form a carbohydrate platform for bacterial detection. Very importantly, this interface can be used for label-free and reagentless detection, both by electrochemical and Quartz Crystal Microbalance (QCM) transducers and by using the direct pili-mannose binding as well as Concanavalin A (Con A) mediated lipopolysaccharides (LPS)-mannose binding. The conductive polymer's unique collective properties are very sensitive to very minor perturbations, which result in significant changes of electrical conductivity and providing amplified sensitivity and improved limits of detection (i.e., 25 cell/mL for electrochemical sensor and 50 cells/mL for QCM sensor), a widened logarithmic range of detection (i.e., 3-7 for pili-mannose binding and 2-8 for Con A mediated binding), high specificity and selectivity, and an extraordinary reliability by a mechanism of internal validation. With these analytical performances, the described biosensor is envisaged for being capable of differentiating Gram-negative bacterial strain and species, for many important applications. [ABSTRACT FROM AUTHOR]

A common goal for astrobiology is to detect organic materials that may indicate the presence of life. However, organic materials alone may not be representative of currently living systems. Thus, it would be valuable to have a method with which to determine the health of living materials. Here, we present progress toward this goal by reporting on the application of laser-induced breakdown spectroscopy (LIBS) to study characteristics of live and dead cells using Escherichia coli ( E. coli) strain K12 cells as a model organism since its growth and death in the laboratory are well understood. Our goal is to determine whether LIBS, in its femto- and/or nanosecond forms, could ascertain the state of a living organism. E. coli strain K12 cells were grown, collected, and exposed to one of two types of inactivation treatments: autoclaving and sonication. Cells were also kept alive as a control. We found that LIBS yields key information that allows for the discrimination of live and dead E. coli bacteria based on ionic shifts reflective of cell membrane integrity. Key Words: E. coli-Trace elements-Live and dead cells-Laser-induced breakdown spectroscopy-Atomic force microscopy. Astrobiology 15, 144-153. [ABSTRACT FROM AUTHOR]

Label-free immunosensors are well suited for detection of microorganisms because of their fast response and reasonable sensitivity comparable to infection doses of common pathogens. Active (lever oscillator and frequency counter) and passive (impedance analyzer) modes of quartz crystal microbalance (QCM) were used and compared for rapid detection of three strains of E. coli. Different approaches for antibody immobilization were compared, the immobilization of reduced antibody using Sulfo-SMCC was most effective achieving the limit of detection (LOD) 8 x 104 CFU·mL-1 in 10 min. For the passive mode, software evaluating impedance characteristics in real-time was developed and used. Almost the same results were achieved using both active and passive modes confirming that the sensor properties are not limited by the frequency evaluation method but mainly by affinity of the antibody. Furthermore, reference measurements were done using surface plasmon resonance. Effect of condition of cells on signal was observed showing that cells ruptured by ultrasonication provided slightly higher signal changes than intact microbes. [ABSTRACT FROM AUTHOR]

A combined capture and detection method comprising of nano-immunomagnetic separation (NIMS) and surface enhanced Raman spectroscopy (SERS) was developed to detect Escherichia coli O157 from liquid media including apple juice. The capture antibodies (cAbs) were immobilized on magnetite–gold (Fe 3 O 4 /Au) magnetic nanoparticles (MNPs) which were used for separation and concentration of the E. coli O157 cells from model liquid food matrix. The capture efficiency (CE) for E. coli O157 using MNP was found to be approximately 84–94%. No cross reactivity was observed with background non-target organisms. There was a significant difference in the mean CE of bacteria captured by MNP and commercially sourced immunomagnetic microbeads (p < 0.05). For the detection of target pathogen, SERS labels were prepared by conjugating gold nanoparticles with Raman reporter molecules and the detector antibody (dAb). Au-Raman label-dAb was interacted with gold coated MNP-cAb- E. coli O157 complex. The ability of this immunoassay to detect E. coli O157 in apple juice was investigated. We have successfully applied the synthesized Fe 3 O 4 /Au nanoclusters to E. coli O157 detection in apple juice using the SERS method. The lowest detectable bacterial cell concentration in apple juice was 10 2 CFU/mL with a total analysis time of less than an hour. This method presents a convenient way of preconcentration, separation, and detection of low levels of target pathogen from liquid food matrix. [ABSTRACT FROM AUTHOR]

Sublethal injury (SI) poses major public health concerns since injured cells are responsible for serious limitations in food diagnostics and are susceptible to recovery, often developing adaptive stress responses. Detection of SI is based on the difference in plate counts between non-selective media, which represent the total cell population, and selective media, to which injured cells become sensitive. Selective media for detection of sublethal membrane damage are often based on NaCl supplement, although there is a lack of consensus in the literature about appropriate levels. Planktonic cells are generally used to investigate SI mechanisms, although they often exhibit different stress tolerance than cell colonies in/on solid food (model) systems. In this work, the effect of growth morphology, colony size and concentration of the gelling agent in the growth media, on the maximum non-inhibitory NaCl concentration in the plating medium was assessed for Escherichia coli , Salmonella Typhimurium and Listeria innocua . Stationary phase cultures of planktonic cells and large and small colonies grown in either 1.5% (w/v) xanthan gum-based system or 2.5% (w/v) xanthan gum-based system exhibited significantly different viable counts and osmotolerance. The effect of cell arrangement and xanthan gum percentage in the growth media depended on the microorganism under investigation. Additionally, differences in the maximum non-inhibitory concentration were evident, with 5.0% (w/v) NaCl for the Gram-negative bacteria and 6.5% (w/v), for L. innocua . Different extent of colony shrinkage and morphological damage was observed as NaCl concentration in the plating medium increased. This information will contribute to define NaCl-based selective media for accurate SI detection under realistic scenarios. [ABSTRACT FROM AUTHOR]

The rise of extended-spectrum beta-lactamase ( ESBL)-producing Escherichia coli in food-producing animals is a growing concern for public health. We investigated ESBL producers isolated from broiler chickens in Brazil and characterized 19 CTX-M-2-producing E. coli. The IS CR1 was detected upstream of the chromosome-located gene blaCTX-M-2, associated with sul-1 type integron structure. CTX-M-2-producing E. coli exhibited different PFGE-types and phylogenetic groups, showing a non-clonal dissemination. The sequence types found ( ST93, ST155 and ST2309) have been associated with humans and animals worldwide. Herein, we report the chromosomal location of blaCTX-M-2 on E. coli, highlighting the risks of multidrug-resistant bacteria in food-producing animals. [ABSTRACT FROM AUTHOR]

The development of low-cost tests for Escherichia coli is hampered by the expense and limited choice of enzyme substrates. Most chromogenic substrates are required in costly amounts, while fluorogenic substrates require an additional apparatus (e.g., an ultraviolet lamp) to be detected. Herein, we propose an alternative chromogenic substrate, resorufin β-D-glucuronide (REG), which is exceptionally sensitive and may be employed in very small amounts. We show that REG can be produced similarly to other simple glucuronides and should therefore be no more expensive. The compound is used by both healthy and injured E. coli, resulting in a pronounced color change from orange to a bright pink. Because the released dye (resorufin) has a high extinction coefficient, substantially lower amounts are needed than for commercially available substrates. The potential of this substrate is demonstrated by a presence/absence test requiring just 0.1 mg of REG/100 mL of water sample, one hundredth of the quantity needed for common chromogenic substrates, with an estimated bulk cost of ⩽0.1 U.S. cents/test. REG shows promise as a chromogenic substrate for E. coli detection and should be considered in the development of new water tests, especially for low-income settings. [ABSTRACT FROM AUTHOR]

The objective of this study was to investigate the antibacterial effect of caffeine on the behavior and changes in cell morphology ofE. coliO157:H7 in a liquid medium and in skim milk. The inhibitory effect of caffeine at different concentrations was determined by inoculatingE. coliO157:H7 in laboratory medium and skim milk samples. Samples were incubated at 37ºC for 48 h, andE. coliO157:H7 population was enumerated. Our results showed that caffeine significantly (P < 0.05) inhibited the growth ofE. coliO157:H7 in laboratory medium and milk samples. A greater than 3.0 log CFU ml−1inhibition was observed in milk containing 5.0 g/L caffeine within 12 h of incubation. Moreover, using flow cytometry, marked changes in the morphology ofE. coliO157:H7 were also observed. Caffeine has potential as an antimicrobial agent and could be used as an effective natural additive to improve the safety of food products. [ABSTRACT FROM AUTHOR]

Background Cholesterol oxidases are important enzymes for applications such as the analysis of cholesterol in clinical samples, the synthesis of steroid derived drugs and are considered as potential antibacterial drug targets. Results The gene choA encoding a cholesterol oxidase from Chryseobacterium gleum DSM 16776 was cloned into the pQE-30 expression vector and heterologously expressed in Escherichia coli JM109 co-transformed with pRARE2. The N-terminally His-tagged cholesterol oxidase (CgChoA) was assigned to be a monomer in solution by size exclusion chromatography, showed a temperature optimum of 35°C, and a pH optimum at 6.75 using 0.011 M MOPS buffer under the tested conditions. The purified protein showed a maximum activity of 15.5 U/mg. CgChoA showed a Michaelis-Menten kinetic behavior only when the substrate was dissolved in water and taurocholate (apparent Km = 0.5 mM). In addition, the conversion of cholesterol by CgChoA was studied via biocatalytic batches at analytical scale, and cholest-4- en-3-one was confirmed as product by HPLC-MS. Conclusion CgChoA is a true cholesterol oxidase which activity ranges among the high performing described cholesterol oxidases from other organisms. Thus, the enzyme broadens the available toolbox of cholesterol oxidases for e.g. synthetic and biosensing applications. [ABSTRACT FROM AUTHOR]