Detection Of Microbial Pathogens Research Articles

La puce à ADN de reséquençage : un outil rapide pour mieux identifier et comprendre une émergence virale etbactérienne

The introduction of microarray technologies in microbiology has transformed the detection and characterization of microbial pathogens. Microarray-based platforms can be classified into different families according to their characteristics and applications. Resequencing microarrays have several advantages over other technologies for pathogen detection and characterization.

Quartz Crystal Microbalance Based Approach for Food Quality

To overcome food borne illness, food quality and safety are major scientific disciplines including food handling, preparation and storage. Detection of contaminants in the food system is the main concern regarding public health. It is necessary to develop an immunoassay system to detect and control food borne pathogens in contaminated foods before they enter into the human food chain and cause infections. The conventional methods including agar plating, chromatography or immunoassay are tedious and time consuming for detection of food contamination, while biosensors are one option to overcome these problems. There is growing interest towards biosensors technology due to high specificity, convenience and quick response. In the field of food science and technology, biosensors technology for the detection of food borne pathogens is more fascinating to the current researchers. In this contest we introduce, quartz crystal microbalance (QCM) a label free and piezoelectric biosensor for the detection of pathogenic microbes and toxins present in the food. The potential significance of the present review is to provide the implementation of QCM based biosensor for the detection of various analytes in food systems including toxic and nontoxic.

Molecular Methods for Microbiological Quality Control of Meat and Meat Products: A Review

Achieving food safety is a global health goal and the food-borne diseases take a major check on global health. Therefore, detection of microbial pathogens in food is the solution to the prevention and recognition of problems related to health and safety. Conventional and standard bacterial detection methods such as culture and colony counting methods and immunology-based methods may take up to several hours or even a few days to yield a result. Obviously, this is inadequate, and recently many researchers are focusing towards the progress of rapid diagnostic methods. The advent of molecular techniques has led to the development of a diverse array of assay for quality control of meat and meat products. Rapid analysis using DNA hybridization and amplification techniques offer more sensitivity and specificity to get results than culture based methods as well as dramatic reduction in the time to get results. Many methods have also achieved the high level automation, facilitating their application as routine sample screening assays. This review is intended to provide an overview of the molecular methods for microbiological quality control of meat and meat products.

Design and construction of a single tube, quantitative endpoint, LATE-PCR multiplex assay for ventilator-associated pneumonia

The goal of this study was to develop a molecular diagnostic multiplex assay for the quantitative detection of microbial pathogens commonly responsible for ventilator-associated pneumonia (VAP) and their antibiotic resistance using linear-after-the-exponential polymerase chain reaction (LATE-PCR). This multiplex assay was designed for the quantitative detection and identification of pathogen genomic DNA of methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter baumannii, Pseudomonas aeruginosa, plus a control target from Lactococcus lactis. After amplification, the single-stranded amplicons were detected simultaneously in the same closed tube by hybridization to low-temperature molecular beacon probes labelled with four differently coloured fluorophores. The resulting hybrids were then analysed by determining the fluorescence intensity of each of the four fluorophores as a function of temperature. This LATE-PCR single tube multiplex assay generated endpoint fluorescent contours that allowed identification of all microbial pathogens commonly responsible for VAP, including MRSA. The assay was quantitative, identifying the pathogens present in the sample, no matter whether there were as few as 10 or as many 100000 target genomes. This assay is rapid, reliable and sensitive and is ready for preclinical testing using samples recovered from patients suffering from ventilator-associated pneumonia.

Microfluidic Chip-Based Detection and Intraspecies Strain Discrimination of Salmonella Serovars Derived from Whole Blood of Septic Mice

Salmonella is a zoonotic pathogen that poses a considerable public health and economic burden in the United States and worldwide. Resultant human diseases range from enterocolitis to bacteremia to sepsis and are acutely dependent on the particular serovar of Salmonella enterica subsp. enterica, which comprises over 99% of human-pathogenic S. enterica isolates. Point-of-care methods for detection and strain discrimination of Salmonella serovars would thus have considerable benefit to medical, veterinary, and field applications that safeguard public health and reduce industry-associated losses. Here we describe a single, disposable microfluidic chip that supports isothermal amplification and sequence-specific detection and discrimination of Salmonella serovars derived from whole blood of septic mice. The integrated microfluidic electrochemical DNA (IMED) chip consists of an amplification chamber that supports loop-mediated isothermal amplification (LAMP), a rapid, single-temperature amplification method as an alternative to PCR that offers advantages in terms of sensitivity, reaction speed, and amplicon yield. The amplification chamber is connected via a microchannel to a detection chamber containing a reagentless, multiplexed (here biplex) sensing array for sequence-specific electrochemical DNA (E-DNA) detection of the LAMP products. Validation of the IMED device was assessed by the detection and discrimination of S. enterica subsp. enterica serovars Typhimurium and Choleraesuis, the causative agents of enterocolitis and sepsis in humans, respectively. IMED chips conferred rapid (under 2 h) detection and discrimination of these strains at clinically relevant levels (<1,000 CFU/ml) from whole, unprocessed blood collected from septic animals. The IMED-based chip assay shows considerable promise as a rapid, inexpensive, and portable point-of-care diagnostic platform for the detection and strain-specific discrimination of microbial pathogens.

Species, developmental stage and infection with microbial pathogens of engorged ticks removed from dogs and questing ticks

Research into tick-borne diseases implies vector sampling and the detection and identification of microbial pathogens. Ticks were collected simultaneously from dogs that had been exposed to tick bites and by flagging the ground in the area in which the dogs had been exposed. In total, 200 ticks were sampled, of which 104 came from dogs and 96 were collected by flagging. These ticks were subsequently examined for DNA of Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Rickettsia spp. and Babesia canis. A mixed sample of adult ticks and nymphs of Ixodes ricinus (Ixodida: Ixodidae) and Haemaphysalis concinna (Ixodida: Ixodidae) was obtained by flagging. Female I. ricinus and adult Dermacentor reticulatus (Ixodida: Ixodidae) ticks dominated the engorged ticks removed from dogs. Rickettsia spp. were detected in 17.0% of the examined ticks, A. phagocytophilum in 3.5%, B. canis in 1.5%, and B. burgdorferi s.l. in 16.0%. Ticks with multiple infections were found only among the flagging sample. The ticks removed from the dogs included 22 infected ticks, whereas the flagging sample included 44 infected ticks. The results showed that the method for collecting ticks influences the species composition of the sample and enables the detection of a different pattern of pathogens. Sampling strategies should be taken into consideration when interpreting studies on tick-borne pathogens.

A polymer microfluidic chip for quantitative detection of multiple water- and foodborne pathogens using real-time fluorogenic loop-mediated isothermal amplification

Inexpensive, portable, and easy-to-use devices for rapid detection of microbial pathogens are needed to ensure safety of water and food. In this study, a disposable polymer microfluidic chip for quantitative detection of multiple pathogens using isothermal nucleic acid amplification was developed. The chip contains an array of 15 interconnected reaction wells with dehydrated primers for loop-mediated isothermal amplification (LAMP), and requires only a single pipetting step for dispensing of sample. To improve robustness of loading and amplification, hydrophobic air vents and microvalves were monolithically integrated in the multi-layered structure of the chip using an inexpensive knife plotter. For quantification, LAMP was performed with a highly fluorescent DNA binding dye (SYTO-82) and the reactions monitored in real-time using a low-cost fluorescence imaging system previously developed by our group (Ahmad et al., Biomed. Microdevices 13(5), 929-937). Starting from genomic DNA mixtures, the chip was successfully evaluated for rapid analysis of multiple virulence and marker genes of Salmonella, Campylobacter jejuni, Shigella, and Vibrio cholerae, enabling detection and quantification of 10-100 genomes per μl in less than 20 min. It is anticipated that the microfluidic chip, along with the real-time imaging system, may be a key enabling technology for developing inexpensive and portable systems for on-site screening of multiple pathogens relevant to food and water safety.

Siderophore-based detection of Fe(iii) and microbial pathogens

Siderophores are low-molecular-weight iron chelators that are produced and exported by bacteria, fungi and plants during periods of nutrient deprivation. The structures, biosynthetic logic, and coordination chemistry of these molecules have fascinated chemists for decades. Studies of such fundamental phenomena guide the use of siderophores and siderophore conjugates in a variety of medicinal applications that include iron-chelation therapies and drug delivery. Sensing applications constitute another important facet of siderophore-based technologies. The high affinities of siderophores for both ferric ions and siderophore receptors, proteins expressed on the cell surface that are required for ferric siderophore import, indicate that these small molecules may be employed for the selective capture of metal ions, proteins, and live bacteria. This minireview summaries progress in methods that utilize native bacterial and fungal siderophore scaffolds for the detection of Fe(III) or microbial pathogens.

A Rolling Stone Gathers No Moss, but Resistant Plants Must Gather Their MOSes

The detection of pathogenic microbes by plant resistance (R) proteins and the subsequent activation of R protein-mediated immunity constitute an important layer in the plant innate immune system. Most R genes encode proteins with nucleotide-binding (NB) and leucine-rich repeat (LRR) domains. The autoimmune mutant suppressor of npr1, constitutive 1 (snc1), that constitutively activates resistance signaling, is a unique model used in our laboratory to dissect the details of TIR (Toll/Interleukin1 receptor)-NB-LRR, protein-mediated defense responses. Suppressor screens of snc1 yielded 15 modifier of snc1 (mos) complementation groups containing second-site mutations, and resulted in the identification of 13 novel MOS genes via either positional cloning or T-DNA tagging. Characterizations of the mos mutants have revealed important roles for transcriptional regulation, RNA processing, protein modifications, and nucleocytoplasmic trafficking in R protein-mediated immunity. The MOS genes have taught us a great deal about the complex mechanisms surrounding R protein activation. Future in-depth genetic and biochemical analyses will further enhance our knowledge of how R proteins are deliberately activated and how specific, targeted immunity is achieved in plants.

Design and Construction of a Single-Tube, LATE-PCR, Multiplex Endpoint Assay with Lights-On/Lights-Off Probes for the Detection of Pathogens Associated with Sepsis

Aims. The goal of this study was to construct a single tube molecular diagnostic multiplex assay for the detection of microbial pathogens commonly associated with septicemia, using LATE-PCR and Lights-On/Lights-Off probe technology. Methods and Results. The assay described here identified pathogens associated with sepsis by amplification and analysis of the 16S ribosomal DNA gene sequence for bacteria and specific gene sequences for fungi. A sequence from an unidentified gene in Lactococcus lactis subsp. cremoris served as a positive control for assay function. LATE-PCR was used to generate single-stranded amplicons that were then analyzed at endpoint over a wide temperature range in a specific fluorescent color. Each bacterial target was identified by its pattern of hybridization to Lights-On/Lights-Off probes derived from molecular beacons. Complex mixtures of targets were also detected. Conclusions. All microbial targets were identified in samples containing low starting copy numbers of pathogen genomic DNA, both as individual targets and in complex mixtures. Significance and Impact of the Study. This assay uses new technology to achieve an advance in the field of molecular diagnostics: a single-tube multiplex assay for identification of pathogens commonly associated with sepsis.

A Resistance Based Biosensor That Utilizes Conductive Microfibers for Microbial Pathogen Detection

Escherichia coli O157:H7 (E. coli O157:H7) is one of the top pathogens of interest for the development of rapid diagnostic systems for food and water samples. The objective of this research is to develop a rapid, novel electrochemical biosensor based on the use of polypropylene microfiber membranes coated with a conductive polypyrrole and antibody functionalized for the biological capture and detection of E. coli O157:H7 inthe field. Using glutaraldehyde, pathogen specific antibodies are covalently attached to conductive microfiber membranes which are then blocked using a 5% bovine serum albumin solution. The functionalized membranes are then exposed to E. coli O157:H7 cells washed in Butterfield’s phosphate buffer and added to a phosphate-buffer electrolyte solution. When a voltage is applied to the system, the presence of the captured pathogen on the fiber surface results in an increase in resistance at the electrotextile electrode surface, indicating a positive result. In this study, the initial resistance of the membrane in the electrochemical system was established and found to range between 5.8 and 13 . The resistance of the system not associated with the electrotextile fibers was calculated to contribute to only 2.8% of the total system resistance, and found not to be significant. A proof of concept experiment was conducted and determined that the electrotextile electrode was able to differentiate between small changes in a solution’s conductivity associated with the presence of E. coli O157:H7 cells over a concentration range of log 0 - 9 CFU/mL.

Detection of Clostridium tetani by fluorescent amplification based specific hybridization

The etiologic agent of tetanus, Clostridium tetani, is an obligate anaerobic and spore-forming bacillus; and remains an important public health problem in developing countries. Although, the diagnosis is based mainly on history and presentation of tetanus symptoms. Some recent reports indicate that a patient may not manifest any symptoms of localized or generalized tetanus. However, one of the new approaches for accurate and rapid detection of microbial pathogens is a polymerase chain reaction (PCR) followed by fluorescence detection. In this report, we have described a fluorescent amplification based specific hybridization (FLASH) based PCR assay for detection of C. tetani. The results of the present study indicated that the FLASH based PCR assay appear to be a suitable and rapid method for identification of cultured isolates of C. tetani. Key words: Clostridium tetani, molecular diagnosis, fluorescent amplification based specific hybridization (FLASH) polymerase chain reaction (PCR), fluorescent probe.

Comparison of conventional culture with SeptiFast real-time PCR for microbial pathogen detection in clinical specimens other than blood

Early detection of aetiological agents is pivotal for adequate therapy for bacterial infections. Although culture is still considered the mainstay for laboratory diagnosis, it often lacks sensitivity, especially in patients already treated with antibiotics. The present study investigated the potential clinical utility of the commercial real-time-PCR-based system SeptiFast (SF), originally intended for diagnosis of sepsis from blood specimens, in the aetiological diagnosis of other bacterial infections, in patients undergoing antibiotic therapy. A total of 53 non-blood specimens were analysed for microbial pathogen detection by conventional culture and with SF real-time PCR: 19 (35.8%) synovial fluids, 9 (17.0%) cardiac valve tissues and 25 (47.2%) purulent exudates from various body sites. Overall, the number of specimens positive for a pathogen by SF (26/53; 49.1%) was significantly greater (P=0.001) than that of specimens positive by culture (10/53; 18.9%). In particular, SF was superior to culture for pathogen detection in cardiac valve tissues and synovial fluids. The analysis of concordance showed a fair agreement between the two methods (kappa value=0.314; 95% confidence interval=0.531-0.097). Even with the limitation of the low number of specimens, this study confirmed the great potential of diagnosing bacterial infections by a molecular approach, and indicates that the real-time PCR SF system can be used for specimens other than blood, from patients undergoing antibiotic treatment.

Evaluation of five DNA extraction methods in the detection of &lt;i&gt;Salmonella enterica&lt;/i&gt; from meat using nested PCR

Polymerase Chain Reaction (PCR) based detection methods have received significant attention in food borne microbial pathogen detection. However, reliability and sensitivity of these methods are highly depending on the extraction of adequate amount of pure DNA using appropriate extraction method. Hence, selection of appropriate DNA extraction method is very important in PCR based detection of microbial pathogens. In this study, the extraction efficiency of five commonly used DNA extraction methods was evaluated. Salmonella enterica was used as experimental organism and five extraction methods were tested for their ability to extract DNA from spiked pork meat samples. Pork meat samples were incubated for four hours after being added a dilution series (10 0 - 10 3 CFU/mL) of Salmonella enterica culture. Then DNA was extracted from those samples by the five commonly used DNA extraction methods. Using extracted DNA, fliC gene of Salmonella was amplified by Nested PCR. Out of those five methods, the modified Fontana and Kapperd methods were found to be more effective in DNA extraction. In addition, those methods gave high detection sensitivity of 10 1 CFU/mL in Nested PCR amplification. Keywords : DNA extraction; Nested PCR; Salmonella, Fontana, Kapperd DOI: http://dx.doi.org/10.4038/jas.v6i1.3809 The Journal of Agricultural Sciences , 2011 Vol.6(1) 24-31

Dead-end ultrafiltration concentration and IMS/ATP-bioluminescence detection of Escherichia coli O157:H7 in recreational water and produce wash

The purpose of this study was to develop a detection method for viable E. coli O157:H7 in fresh produce and recreational water. The method was evaluated using eight samples of produce wash and recreational water with or without spiked E. coli O157:H7 at ≤102CFU·ml−1 and concentrated using dead-end ultrafiltration (DEUF) to produce primary and secondary retentates. Fifty-four matrix replicates of undiluted secondary retentates or dilutions (1:2 or 1:10 in buffer) were evaluated using an IMS/ATP bioluminescence assay (IMS/ATP). Combining primary and secondary DEUF yielded a 2–4 log10 increase in E. coli O157:H7 concentrations in spiked samples and resulted in signal-to-noise ratios 2–219 fold higher than controls, depending on the sample type. DEUF increased the concentration of E. coli O157:H7 to within the detectable limits of IMS/ATP. The combined assay provided detection of viable E. coli O157:H7 in produce and recreational water. Accurate detection of microbial pathogens using DEUF and IMS/ATP could reduce disease outbreaks from contaminated water sources and food products.

Review: Norovirus prevalence in Belgian, Canadian and French fresh produce: A threat to human health?

Foodborne viruses, especially noroviruses (NoV), are increasingly reported as the cause of foodborne outbreaks. NoV outbreaks have been reported linked to fresh soft red fruits and leafy greens. Belgium, Canada and France were the first countries to provide data about the prevalence of NoV on fresh produce. In total, 867 samples of leafy greens, 180 samples of fresh soft red fruits and 57 samples of other types of fresh produce (tomatoes, cucumber and fruit salads) were analyzed. Firstly, the NoV detection methodology, including virus and RNA extraction, real-time RT-PCR and quality controls were compared among the three countries. In addition, confirmation and genotyping of the NoV strains was attempted for a subset of NoV positive samples using conventional RT-PCR targeting an alternative region followed by sequencing. Analysis of the process control showed that 653, 179 and 18 samples of the leafy greens, soft red fruits and other fresh produce types were valid for analysis based on the recovery of the process control. NoV was detected by real-time RT-PCR in 28.2% (N=641), 33.3% (N=6) and 50% (N=6) of leafy greens tested in Canada, Belgium and France, respectively. Soft red fruits were found positive by real-time RT-PCR in 34.5% (N=29) and 6.7% (N=150) of the samples tested in Belgium and France, respectively. 55.5% (N=18) of the other fresh produce types, analyzed in Belgium, were found NoV positive by real-time RT-PCR. Conventional RT-PCR resulted in an amplicon of the expected size in 19.5% (52/266) of the NoV positive samples where this assay was attempted. Subsequent sequencing was only successful in 34.6% (18/52) of the suspected amplicons obtained by conventional RT-PCR. From this study, using the described methodology, NoV genomes were frequently detected in fresh produce however sequence confirmation was not successful for the majority of the samples tested. Infection or outbreaks were rarely or not known to be related to the NoV positive samples. With the increase in sensitivity of the detection methodology, there is an increasing concern about the interpretation of positive NoV results by real-time amplification. Strategies to confirm the results by real-time RT-PCR should be developed in analogy with the detection of microbial pathogens in foods. Detection might indicate contact with NoV in the fresh produce chain. Consequently, a potential risk for infection cannot be excluded but the actual risk from RT-PCR NoV positive produce is still unknown. Studies should be designed determining the probability of infection related to the presence or levels of NoV genomic copies.

Editorial comment

Editorial comment

High Throughput Multiplex Assay for Species Identification of Papua New Guinea Malaria Vectors: Members of the Anopheles punctulatus (Diptera: Culicidae) Species Group

Malaria and filariasis are transmitted in the Southwest Pacific region by Anopheles punctulatus sibling species including An. punctulatus, An. koliensis, the An. farauti complex 1-8 (includes An. hinesorum [An. farauti 2], An. torresiensis [An. farauti 3]). Distinguishing these species from each other requires molecular diagnostic methods. We developed a multiplex polymerase chain reaction (PCR)-based assay specific for known species-specific nucleotide differences in the internal transcribed spacer 2 region and identified the five species most frequently implicated in transmitting disease (An. punctulatus, An. koliensis, An. farauti 1, An. hinesorum, and An. farauti 4). A set of 340 individual mosquitoes obtained from seven Papua New Guinea provinces representing a variety of habitats were analyzed by using this multiplex assay. Concordance between molecular and morphological diagnosis was 56.4% for An. punctulatus, 85.3% for An. koliensis, and 88.9% for An. farauti. Among 158 mosquitoes morphologically designated as An. farauti, 33 were re-classified by PCR as An. punctulatus, 4 as An. koliensis, 26 as An. farauti 1, 49 as An. hinesorum, and 46 as An. farauti 4. Misclassification results from variable coloration of the proboscis and overlap of An. punctulatus, An. koliensis, the An. farauti 4. This multiplex technology enables further mosquito strain identification and simultaneous detection of microbial pathogens.

Visual optical discrimination and detection of microbial pathogens based on diverse interactions of conjugated polyelectrolytes with cells

Pathogen infections caused by Candida albicans (C. albicans) and Escherichia coli (E. coli) have led to extremely serious consequences. Cultures of blood samples, which are regarded as the diagnosis standard, often exhibit variable sensitivity and always take several days to detect certain pathogens. Here we demonstrate for the first time that a blend of two cationic conjugated polymers, between which fluorescence resonance energy transfer (FRET) can be induced, is used to detect and discriminate C. albicans and E. coli in a rapid, simple and visual way without the need for any complicated instrumentation. Coupled with general polymerase chain reaction (PCR) technology, the method can detect pathogens specifically independent of electrophoresis analyses and real-time PCR, which avoids utilizing detrimental nucleic dyes and expensive fluorophore-labeled primers. Our new cationic conjugated polymer blend has two unique features. Firstly, the assay is simple and rapid. Upon mixing fungi or bacteria with the polymer blend, one can trigger the signal response directly in solution using a fluorimeter, which may have a far-reaching impact on clinical diagnosis. Secondly, the most important characteristic of the assay is direct visualization under UV light, which makes it more convenient than other methods that require sophisticated instruments. The cationic conjugated polymer blend exhibits great potential as an ideal diagnostic agent for the identification of pathogens.

Application of FTA®Cards to Sample Microbial Plant Pathogens for PCR and RT‐PCR

AbstractFor the detection of microbial plant pathogens, like fungi, bacteria, viruses and viroids, methods based on nucleic acids have gained importance as the availability of sequence information increased. This requires well‐established extraction procedures that are cheap, non‐laborious, safe and reliable. The paper cards introduced by Flinders Technology Associates, acronym FTA®cards, offer a simple tool to sample and preserve nucleic acids from many kinds of biological specimen and have been already tested for their potential to sample and process several plant pathogens in PCR and RT‐PCR. We have tested FTA cards for the sample preparation of a broader range of plant pathogens with different NA contents and subsequent amplification by PCR, RT‐PCR as well as multiplex PCR.