Method For Detection Of Bacteria Research Articles

Fluorescence Imaging of E. coli using CdSe Quantum Dots

Rapid and reliable method for the selective detection of pathogenic bacteria is critical in environmental monitoring, disease control, food safety and for the diagnosis of infectious diseases. In this study, we have synthesized hydrophilic fluorescent CdSe Quantum dots (CDSe QDs) and characterized using UV-Vis, IR and fluorescence spectroscopy. Crystallinity of the synthesized Qdots were analysed using XRD. As synthesized CdSe QDs were used for the imaging of buccal epithelial cells and pathogenic bacteria. Selectivity of CdSe QDs to detect E. coli was improved by the functionalization of QDs with glycerol. Easy imaging steps and decreased imaging time with the current procedure shows the potential for biomedical imaging.

GC-IMS headspace analyses allow early recognition of bacterial growth and rapid pathogen differentiation in standard blood cultures.

Outcome of patients with blood stream infections (BSI) depends on the rapid initiation of adequate antibiotic therapy, which relies on the fast and reliable identification of the underlying pathogen. Blood cultures (BC) using CO2-sensitive colorimetric indicators and subsequent microbiological culturing are the diagnostic gold standard but turnaround times range between 24 and 48 h. The detection of volatile organic compounds of microbial origin (mVOC) has been described as a feasible method for identifying microbial growth and to differentiate between several microbial species. In this study, we aimed to investigate the ability of mVOC analyses using a gas chromatograph coupled to an ion mobility spectrometer (GC-IMS) for the recognition of bacterial growth and bacterial differentiation in BCs. Therefore, samples of whole blood and diluted bacterial suspension were injected into aerobic and anaerobic BC bottles and incubated for 8 h. Headspace samples from cultures of Escherichia coli (DSM 25944), Staphylococcus aureus (DSM 13661), and Pseudomonas aeruginosa (DSM 1117) were investigated hourly and we determined at which point of time a differentiation between the bacteria was possible. We found specific mVOC signals in the headspace over growing BCs of all three bacterial species. GC-IMS headspace analyses allowed faster recognition of bacterial growth than the colorimetric indicator of the BCs. A differentiation between the three investigated species was possible after 6 h of incubation with a high reliability in the principal component analysis. We concluded that GC-IMS headspace analyses could be a helpful method for the rapid detection and identification of bacteria in BSI.

Wild Bonelli’s eagles (Aquila fasciata) as carrier of antimicrobial resistant Salmonella and Campylobacter in Eastern Spain

Wild birds have repeatedly been found to be involved in the dissemination of enteric bacterial pathogens in the environment. The aim of this study was to determine the occurrence of Salmonella and Campylobacter as well as the antimicrobial resistance in wild Bonelli’s eagles nestlings in Eastern Spain. In addition, we compared the efficiency of two sampling methods (fresh faecal samples from nest and cloacal swabs from nestlings) for detection of both bacteria. A total of 28 nests with 45 nestlings were analysed. In the nest, Salmonella occurrence was 61 ± 9.2%, while Campylobacter occurrence was 11 ± 5.8% (p < 0.05). In the nestlings, Salmonella occurrence was 36 ± 7.1%, while Campylobacter occurrence was 11 ± 4.7% (p < 0.05). Eight Salmonella serovars were identified, and the most frequently isolated were S. Enteritidis, S. Typhimurium, S. Houston, and S. Cerro. Only one Campylobacter species was identified (C. jejuni). Regarding antimicrobial resistance, the Salmonella strains isolated were found to be most frequently resistant to ampicillin and to tigecycline; however, the sole Campylobacter strain recovered was multidrug resistant. In conclusion, this study demonstrated that wild Bonelli’s eagles nestlings are greater carriers of Salmonella than of Campylobacter. Both Salmonella and Campylobacter isolates exhibited antimicrobial resistance. In addition, faecal samples from nests were most reliable for Salmonella detection, while cloacal swab from nestlings were most reliable for Campylobacter detection.

Dark-Field Microscopic Detection of Bacteria using Bacteriophage-Immobilized SiO2@AuNP Core-Shell Nanoparticles.

To replace molecular biological and immunological methods, biosensors have recently been developed for the rapid and sensitive detection of bacteria. Among a wide variety of biological materials, bacteriophages have received increasing attention as promising alternatives to antibodies in biosensor applications. Thus, we herein present a rapid and highly selective detection method for pathogenic bacteria, which combines dark-field light scattering imaging with a plasmonic biosensor system. The plasmonic biosensor system employs bacteriophages as the biorecognition element and the aggregation-induced light scattering signal of gold nanoparticle-assembled silica nanospheres as a signal transducer. Using Staphylococcus aureus strain SA27 as a model analyte, we demonstrated that the plasmonic biosensor system detects S. aureus in the presence of excess Escherichia coli in a highly selective manner. After the sample and the S. aureus phage S13'-conjugated plasmon scattering probe were mixed, S. aureus detection was completed within 15-20 min with a detection limit of 8 × 104 colony forming units per milliliter.

VALIDATION OF METHOD FOR YERSINIA ENTEROCOLITICA ISOLATION FROM RAW MATERIALS OF ANIMAL ORIGIN

Upward trend in the number of human yersiniosis cases, caused by bacterium Yersinia enterocolitica, is globally observed nowadays. This microorganism is widely spread in the environment, able to persist for prolonged periods in animal products and propagate under low temperatures. Basic infection sources are meat and meat products. In order to isolate Yersinia enterocolitica from food and feed samples horizontal method for the detection pursuant to GOST ISO 10273-2013 was used. It was noted, that Yersinia enterocolitica isolation is associated with certain difficulties, because the sample contains only small quantities of the agent and only the use of special techniques allows removing the concurrent microflora. It was proposed to use cold enrich­ment (4 ± 1) °C of the test material before conventional technique is started. The technique was validated pursuant to GOST ISO 16140-2011. As a result, it was established that validated method for Yersinia enterocolitica bacteria detection in food products, performed at the Microbiology Laboratory, is specific. The method sensitivity is 10 CFU/cm3. Intralaboratory reproducibility and repeatability were confirmed by relevant tests. Additional culture step at (4 ± 1) °C allows complete inhibition of non-psychrophilic microorganisms’ growth.

Fluorometric detection of sulfate-reducing bacteria via the aggregation-induced emission of glutathione-gold(I) complexes.

It is reported that gold(I)-thiolate complexes can display aggregation-induced emission (AIE) similar to organic fluorogens. On addition of lead(II) to glutathione-gold(I) complexes, a supermolecular structure of type GSH-Au(I)-Pb(II) is formed through strong coordination between Pb(II) and GSH. Its fluorescence is quenched by sulfide due to the formation of PbS which destroys the GSH-Au(I)-Pb(II) complex. The finding was used to design a method for fluorometric detection of sulfate-reducing bacteria (SRB) which produce sulfide. The time needed to reduce fluorescence to 10% of its initial intensity linearly dependent on the logarithm of the SRB concentrations in the ranging from 10 to 1 × 10^7 cfu mL-1. The assay time is also reduced down to 4 days even if the SRB concentration is as low as 10 cfu mL-1. Graphical abstract Schematic presentation of aggregation-induced emission (AIE)-active GSH-Au(I) complexes based fluorescence detection of SRB. The GSH-Au(I) complexes turn into aggregation and display strong emissive property in the presence of Pb2+. Then the fluorescence of GSH-Au(I)-Pb(II) complexes can be quenched by S2- generated by SRB.

Quantum Dot-Based F0F1-ATPase Aptasensor for Vibrio parahaemolyticus Detection

Sensitive detection of pathogenic bacteria is of great significance for food safety and public health. This study aimed to report a novel F0F1–ATPase aptasensor based on quantum dots (QDs) for quantitative detection of Vibrio parahaemolyticus. The fluorescent QDs were labeled onto the surface of chromatophores to act as an indicator. Aptamer target to V. parahaemolyticus was linked to the “rotator” e-subunit of F0F1–ATPase within chromatophores to act as a recognition element. On the basis of aptamer and V. parahaemolyticus binding, the loading of e-subunit was increased and the rotation rate of F0F1-ATPase was decreased, resulting in a decrease in the concentration of H+ out of the chromatophores. The increased pH value induced the increased fluorescence intensity of QDs. By using this fluorescent signal strategy in the detection, a limit of detection (LOD) as low as 7 cfu/mL was successfully achieved for ultrasensitive detection of V. parahaemolyticus. This aptasensing assay had no serious cross reaction with interfering species, and was successfully applied to the determination of V. parahaemolyticus in spiked shrimp and salmon samples. The F0F1-ATPase aptasensor provides a promising method for pathogenic bacteria detection in food safety and other application.

Detection of vibrio harveyi using iavh primer in shrimp fry infected under different immersion time

Methods for the early detection of the pathogenic luminous Vibrio bacteria would be beneficial through enabling timely (early) implementation of preventative measures to reduce shrimp mortality due to vibriosis. This study aimed to measure the sensitivity of the IAVh haemolysin primer in detecting pathogenic Vibrio harveyi in tiger shrimp (Penaeus monodon) fry. This research was conducted in the Fish Health and Environment Laboratory at the Research Institute for Coastal Aquaculture (BPPBAP) in Maros, South Sulawesi, Indonesia. In this study, tiger shrimp fry were infected with the pathogenic V. harveyi bacteria through an immersion method at concentrations of 105, 106, and 107CFU/mL. Samples were collected after immersion times of 6 hours, 12 hours, and 24 hours. The presence of the haemolysin gene was detected through the PCR (Polymerase Chain Reaction) technique using the IAVh primer set. Electrophoresis results detected the presence of pathogenic V. harveyi bacteria after 6 hours of exposure, although the DNA bands produced were still thin. After 12 hours exposure, the DNA bands of pathogenic V. harveyi bacteria were clearly visible. However, after 24 hours exposure, the presence of the haemolysin gene of pathogenic V. harveyi bacteria was no longer detected. Similar results were obtained for all bacterial concentrations tested. It is thought that the 24 hour results are due to a decline in the population of V. harveyi bacteria living within the shrimp body and shrimp culture media. These findings indicate that it is important to implement rapid detection methods early on in cases of bacterial infection in shrimp (0 − 12 hours), since it was difficult to detect the presence of pathogenic bacteria in the shrimp body tissues more than 12 hours post infection.

Combination of a flow cytometric bead system with 16S rRNA-targeted oligonucleotide probes for bacteria detection.

Here we report a bacteria detection method based on a flow cytometric bead system and 16S rRNA-targeted oligonucleotide probes. Polymerase chain reaction (PCR) was first used to acquire bacterial DNA including bacteria-specific sequences. Half of the resulting target DNA was then captured by a capture probe immobilized on a magnetic microbead (MB) surface. The other half of the target DNA was hybridized with a fluorescence-labeled signal probe. In this manner, a sandwich DNA hybridization involving a MB-based capture probe, the target DNA, and a signal probe was realized. The MB carriers modified with reporter dye were analyzed one by one by flow cytometry through a capillary. Using PCR amplicons and this flow cytometric bead system, a detection limit of 180cfumL-1 was achieved, along with high selectivity that permitted the discrimination of different targets when challenged with control bacteria targets and multiplexing capabilities that enabled the simultaneous detection of two kinds of bacteria. Given these advantages, the developed method can be used for the highly sensitive and specific PCR amplicon analysis of DNA extracted from a fresh bacterial culture, as well as multiplex target analysis. Graphical abstract The flow cytometric bead system with 16S rRNA-targeted oligonucleotide probes for bacteria detection developed in this work. This system is highly specific and sensitive, with a detection limit of 180cfumL-1 bacteria.

A simple and rapid colorimetric bacteria detection method based on bacterial inhibition of glucose oxidase-catalyzed reaction

Rapid and sensitive detection of live bacteria is crucial in the realm of clinical diagnosis, food industry and environmental quality control. A portable, feasible and cost-effective platform which enables rapid and accurate live bacteria detection is still challenging. Herein, we present a Bacterial Inhibition of GOX-catalyzed Reaction (BIGR) method for rapid and broad-spectrum detection of live bacteria, which results in a visible color change without any complex instrumentations. We validated this strategy with five common clinical bacteria, namely Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Streptococcus mutans and Salmonella pullorum. This method precludes the interference of dead bacteria. Only several microliters of samples and reagents are required in this assay and the overall analysis time is less than 20 min. In a further demonstration, the presented method is successfully applied for detection of ascites samples from infected mice. Our results suggest that this method serves as a rapid and dose-dependent visual detection of pathogens in the clinical and daily life.

PCR Analysis Methods for Detection and Identification of Beer-Spoilage Lactic Acid Bacteria.

Polymerase chain reaction (PCR) analysis enables rapid and accurate detection of beer-spoilage lactic acid bacteria (LAB). Hop resistance genes, horA and horC, are utilized as genetic markers to determine the spoilage ability of LAB strains. PCR analysis of horA and horC, combined with multiplex PCR methods of 12 beer-spoilage species, enables simultaneous and comprehensive detection easily and inexpensively.

Polydimethylsiloxane Gold Nanoparticle Composite Film as Structure for Aptamer-Based Detection of Vibrio parahaemolyticus by Surface-Enhanced Raman Spectroscopy

This paper investigated a new detection method for food pathogenic bacteria Vibrio parahaemolyticus based on surface-enhanced Raman scattering (SERS). Nanostructures consisting of Aptamer-Au nanoparticles-polydimethylsiloxane (Apt-Au-PDMS film) were firstly used as SERS substrate. Aptamers were immobilized on the functionalized PDMS film covered with Au nanoparticles (AuNPs) to act as a capture structure via the affinity binding of aptamer, and Vibrio parahaemolyticus. AuNPs were modified with the aptamer and Raman reporter molecule 4-mercaptobenzoic acid (4-MBA) to act as SERS-sensing probes that bind to the target in the same way as the Apt-Au-PDMS film. The solid sandwich structure was formed by capture structure-target-SERS-sensing probes. The concentration of Vibrio parahaemolyticus can be quantified by measurement of the SERS intensity of 4-MBA. Under optimal conditions, the signal of 4-MBA at 1590 cm−1 was linearly related to the Vibrio parahaemolyticus concentration in the range between 1.2 × 102 and 1.2 × 106 cfu·mL−1. Recoveries ranging from 97.7% to 105.3% were found when analyzing spiked prawn samples. This developed ultrasensitive aptamer-based SERS detection structure suggested that it would be a promising strategy for a variety of sensing applications.

Rapid and sensitive detection of Escherichia coli O157:H7 using coaxial channel-based DNA extraction and microfluidic PCR

In this study, a rapid and sensitive method for detection of Escherichia coli O157:H7 using the coaxial channel-based DNA extraction and the microfluidic PCR was proposed and verified. The magnetic silica beads were first pumped into the coaxial channel, which was captured in the coaxial channel more uniformly by applying the multiring high-gradient magnetic field. After the E. coli O157:H7 cells were lysed with the lysis buffer to release the DNA, the improved coaxial channel was used to efficiently extract the DNA, followed by washing with ethanol to remove the residual proteins and eluting with a small volume of deionized water to obtain the purified and concentrated DNA. Finally, the obtained DNA was amplified and determined using the microfluidic PCR. This proposed bacteria detection method was able to detect E. coli O157:H7 as low as 12 cfu/mL when the large volume (10 mL) of bacterial sample was used, and the recovery of E. coli O157:H7 in the spiked milk samples ranged from 97.4 to 100.6%. This proposed bacteria detection method has shown great potential to detect lower concentration of E. coli O157:H7 from larger volumes of sample.

PO-269 Development of a fluorescence in situ hybridization (FISH) method for detection of intra-tumour bacteria involved in pancreatic cancer chemoresistance

IntroductionPancreatic cancer (PC) is projected to become the second leading cause of cancer-related deaths in 2020. Recent studies demonstrate that bacteria are a component of pancreatic tumour microenvironment, and they may also play a critical role in mediating resistance to chemotherapy.1 Remarkably, a prospective study showed that the presence of two oral pathogens, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, was associated with an elevated risk of developing PC.2Material and methodsThis study aimed at developing a novel fluorescence in situ hybridization (FISH) based method to specifically detect oral bacteria in formalin-fixed paraffin-embedded (FFPE) sections from PC patients. Multi-wavelength confocal spinning disk microscopy was used to discriminate with high spatial resolution and sensitivity the tissue high autofluorescence signal from even low number of emitters/bacteria. Bacterial FISH probes were primers for the 16 s rRNA molecule.Results and discussionsFISH allowed the in situ localisation and the study of spatial organisation of bacterial cells as they occur in PC. FISH results were always definitive and well-detectable both in in 4 and 20 µm FFPE sections. The intensity of the fluorescence was a direct measure for the activity of the bacterial cells, whereas inactive cells were recognised by their low intensity fluorescence. Thus, our method gives a optimal sensitivity of fluorescence and low background by subtracting autofluorescence components.Our FISH preliminary data confirmed the presence of bacteria in the pancreatic tumour microenvironment and support further studies aimed at elucidating how oral bacteria are involved in PC aetiology.It is well-known that the chemoresistance (intrinsic and acquired) has been a major problem for PC treatment3 and a recent study showed the role of bacteria in resistance to gemcitabine.1 To this end, the establishment of our reliable FISH protocol allows us to further investigate the potential role of oral bacteria in the failure of standard treatments for PC.ConclusionOur new robust methodology will contribute to obtain meaningful data and to unravel the complex interaction between oral bacteria and pancreatic cancer chemoresistance in clinically well-annotated tissues, ultimately identifying specific bacteria as potential biomarkers for early detection/treatment.References. Geller, et al. Science2017;357:1156–1160.. Farrell JJ, et al. Gut2012; 61:582–588.. Coppola, et al. Drug Resist. Updates 2017;31:43–51.

Fabrication of Ag@TiO2 electrospinning nanofibrous felts as SERS substrate for direct and sensitive bacterial detection

In order to develop a novel method for bacteria detection, Ag@TiO2 electrospinning Nanofibrous felts have been fabricated by in situ decoration of size tunable Ag nanoparticles on anatase TiO2 nanofibers. The morphologies and structures of the as-prepared Ag@TiO2 electrospinning nanofibrous felts were characterized by techniques of scanning electron microscopy, energy-dispersive X-ray spectroscopy, transmission electron microscopy, and X-ray diffraction. The result show that Ag NPs were uniformly deposited on the surfaces of TiO2 nanofibers, especially when the deposition time in Tollens’ reagent was 10 min. The free-standing composite nanofibrous felts processes superb performance of surface enhanced Raman scattering, which was demonstrated by small probe mecules of 4-mercaptobenzoic acid and 4-mercaptophenol, and the lowest detection limit was near to10−9 mol/L. More importantly, the as-fabricated Ag@TiO2 nanofibrous felts as SERS substrate can detect biomacromolecules such as bacteria of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) directly without previous bacteria-aptamer conjugation as the traditonal process. In addition, the composite nanofibrous felts also show outstanding antimicrobial activities against both Gram-negative of E. coli and Gram-positive of S. aureus, evaluated by method of absorption and microscopic observation. The results show that the bacteriostasis rate is reached to 99%, especially significant for E. coli. The free-standing, high porous, and flexible substrate of Ag@TiO2 electrospun nanofibrous membranes have potential application in versatile fields such as microorganism sensors and water purifier.

Electrodeposition of a magnetic and redox-active chitosan film for capturing and sensing metabolic active bacteria

Rapid and portable detection of viable pathogen is highly desired to minimize the risk of foodborne pathogen outbreaks. Here we report a proof-of-concept fabrication methodology of a multifunctional film that allows established methods from bacterial recognition (antibodies) and nanotechnology (magnetic nanoparticles) to be coupled with electrochemical signal processing methods for detection of viable bacteria. Specifically, we enlist a sequence of externally applied electrical and magnetic signals to: i) guide the self-assembly of stimuli-responsive biopolymer; ii) incorporate magnetic nanoparticles to form a magnetic layer; iii) electro-synthesize a signal processing layer (redox-capacitor). The function of the magnetic layer is collecting and concentrating MMP-bacteria through magnetic attractions between MMP-bacteria and the magnetic layer. The function of the signal processing layer is amplifying electrochemical detection of the collected bacteria by engaging the redox-active mediators with the redox capacitor. Importantly, the fabrication demonstrated here is simple, controllable, and reagentless.

Detection in raw cow's milk of coliform bacteria - reservoir of antibiotic resistance

Antibiotic resistance is one of the main threat for people, especially since food products are more frequently identified as a reservoir of antibiotic resistance genes. Thus, the aim of the study was to analyse the Polish raw cow's milk in terms of the presence of coliform bacteria resistant to antibiotic as well as to identify volatile compounds (VC) characteristic for such bacteria. Further, the identified profile of VC could be used in development of coliform bacteria detection method directly in the product. The obtained data showed that among coliform bacteria strain isolated from Polish raw cow's milk 88% were resistant to penicillin, 39% to kanamycin, 43% to streptomycin, 78% to chloramphenicol and 55% to tetracycline. Moreover, tetM gene, frequently connected with mobile genetic elements, were detected in 42% of isolated coliform strains. Due to widespread of coliform bacteria resistant to antibiotic in raw milk, simple methods of detection of coliform bacteria in such product are required. Performed analysis suggests that lack of methional might be used as an indicator of raw cow's milk contamination with coliform bacteria.

Detection of Pathogenic Microorganisms by Microfluidics Based Analytical Methods.

Microfluidics based biochemical analysis shows distinctive advantages for fast detection of pathogenic microorganisms. This Feature summarizes the progress in the past decade on microfluidic methods for purification and detection of pathogenic bacteria and viruses as well as their applications in food safety control, environmental monitoring, and clinical diagnosis.

Indoor bacteria and asthma in adults: a multicentre case-control study within ECRHS II.

Both protective and adverse effects of indoor microbial exposure on asthma have been reported, but mostly in children. To date, no study in adults has used non-targeted methods for detection of indoor bacteria followed by quantitative confirmation.A cross-sectional study of 198 asthmatic and 199 controls was conducted within the European Community Respiratory Health Survey (ECRHS) II. DNA was extracted from mattress dust for bacterial analysis using denaturing gradient gel electrophoresis (DGGE). Selected bands were sequenced and associations with asthma confirmed with four quantitative PCR (qPCR) assays.15 out of 37 bands detected with DGGE, which had at least a suggestive association (p<0.25) with asthma, were sequenced. Of the four targeted qPCRs, Clostridium cluster XI confirmed the protective association with asthma. The association was dose dependent (aOR 0.43 (95% CI 0.22-0.84) for the fourth versus first quartile, p for trend 0.009) and independent of other microbial markers. Few significant associations were observed for the three other qPCRs used.In this large international study, the level of Clostridium cluster XI was independently associated with a lower risk of prevalent asthma. Results suggest the importance of environmental bacteria also in adult asthma, but need to be confirmed in future studies.

A simple, rapid and cost-effective colorimetric assay based on the 4-mercaptophenylboronic acid functionalized silver nanoparticles for bacteria monitoring

The rapid and cost- effective detection of bacteria is of great importance in various fields. Herein, a novel colorimetric detection method for Gram-negative bacteria based on the inhibition of the aggregation of 4-mercaptophenylboronic acid functionalized AgNPs (MPBA-AgNPs) was demonstrated. MPBA, one of the boronic acid derivative, which could react with the cis-diol groups contained in saccharides on the bacteria cells, was used as the recognition molecule for bacteria detection. The aggregation of MPBA-AgNPs could be inhibited by bacterial cells, resulting in different color change, which could be distinguished with UV–vis spectrometer and the naked eye. The concentration of E. coli, a model of Gram-negative bacteria, could be determined in a dynamic range from 5 × 104 cfu mL−1 to 1 × 107 cfu mL−1 with a LOD of 0.9 × 104 cfu mL−1 with fast response of 20 min. Meanwhile, a smartphone based measuring analysis was established to make it suitable for routine laboratory applications. Moreover, the concept of the MPBA-AgNPs based colorimetric strategy could be expanded to other Gram-negative bacteria suggesting a universal platform for bacterial assay.