Bacterial Pathogenesis: Methods and Protocols

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Plos Medicine 3 , e Multiplex PCR assay for detection of Actinobacillus pleuropneumoniae, Pasteurella multocida and Haemophilus parasuis in lungs of pigs from a slaughterhouse. Folia microbiologica 55 , — Kaper, J. Pathogenic Escherichia coli. Nature Review. Nature Reviews Microbiology 2 , — Mikkelsen, L. Horiguchi, Y. Curr Top Microbiol Immunol , — Brooke, C. Erysipelothrix rhusiopathiae: bacteriology, epidemiology and clinical manifestations of an occupational pathogen. Journal of Medical Microbiology 48 , — Gottschalk, M.

Foodborne Bacterial Pathogens door Arnaud Bridier (Boek) -

Canada: Distribution of Streptococcus suis from to and Actinobacillus pleuropneumoniae from to serotypes isolated from diseased pigs. Canadian Veterinary Journal-revue Veterinaire Canadienne 56 , — Hu, X. Occurrence and source analysis of typical veterinary antibiotics in manure, soil, vegetables and groundwater from organic vegetable bases, northern China. Environmental Pollution , — Ling, Z. Residues of veterinary antibiotics in manures from feedlot livestock in eight provinces of China.

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Bacterial Pathogenesis, Part A: Identification and Regulation of Virulence Factors, Volume 235

Moorkamp, L. Detection of respiratory pathogens in porcine lung tissue and lavage fluid. Veterinary Journal , — Lee, K. Cowart, R. Pasteurella multocida and Bordetella bronchiseptica in atrophic rhinitis and pneumonia in swine. Navacharoen, N. Hearing and vestibular loss in Streptococcus suis infection from swine and traditional raw pork exposure in northern Thailand. Hill, J. Biochemical analysis, cpn 60 and 16S rDNA sequence data indicate that Streptococcus suis serotypes 32 and 34, isolated from pigs, are Streptococcus orisratti.

Veterinary microbiology , 63—69 Kielstein, P. Designation of 15 serovars of Haemophilus parasuis on the basis of immunodiffusion using heat-stable antigen extracts. Journal of clinical microbiology 30 , — Jiang, X. Live Streptococcus suis type 5 strain XS provides cross-protection against infection by strains of types 2 and 9. Vaccine 34 Bak, H. Protection of vaccinated pigs against experimental infections with homologous and heterologous Hoemophilus parasuis. Veterinary Record , — Wei, Z. Characterization of Streptococcus suis isolates from the diseased pigs in China between and Wang, J.

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Investigation of antimicrobial resistance and genetic correlation analysis of Haemophilus parasuis isolated from Guangdong Province. Chinese Veterinary Science Hu, P. Comparative genomics study of multi-drug-resistance mechanisms in the antibiotic-resistant Streptococcus suis R61 strain. PLoS One 6 , e Okwumabua, O. A polymerase chain reaction PCR assay specific for Streptococcus suis based on the gene encoding the glutamate dehydrogenase. Fems Microbiology Letters , 79—84 Oliveira, S. Journal of Veterinary Diagnostic Investigation 13 , — Nagai, S.

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Detection of Bordetella bronchiseptica by the polymerase chain reaction.

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Research in Microbiology , — Rapid identification of Salmonella serovars in feces by specific detection of virulence genes, invA and spvC, by an enrichment broth culture-multiplex PCR combination assay. Journal of clinical microbiology 34 , — Shimoji, Y. Journal of clinical microbiology 36 , 86 Download references. Correspondence to Qigai He. Reprints and Permissions. By submitting a comment you agree to abide by our Terms and Community Guidelines.

If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Advanced search. Skip to main content. Subjects Bacterial pathogenesis Cellular microbiology. Abstract Bacterial diseases of swine are a kind of multifactorial and uncontrollable diseases that commonly exist in pig farms all over the world and will lead to huge economic losses every year. Introduction Bacterial diseases heavily affect the health of swine, especially, respiratory system and digestive system diseases which are reported to be associated with intensive pig production 1.

Materials and Methods Samples collection All pig tissue samples were collected from 16 Chinese major pig breeding provinces from to and delivered to the Animal Disease Diagnostic Center of Huazhong Agricultural University for identification of bacterial pathogens, which is a reference lab in China where people send pig samples for diagnosis Fig. Figure 1. Full size image.

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Table 1 Samples information. For food matrices with different properties, various strategies have been developed Stevens and Jaykus ; Bhunia Here we will briefly highlight a few commonly used methods from recent studies. Centrifugation and filtration are still among the most commonly used physical separation methods due to the simplicity of these techniques. When using centrifugation and filtration, as series of concentration steps are usually used, the recovery may decrease during multiple separations and repeated washings.

While many chemical methods and physicochemical methods have been investigated in foodborne separation and concentration Stevens and Jaykus , methods based on biological interactions such as immunomagnetic separation IMS has become one major approach for separating foodborne bacteria followed by various detection methods Uyttendaele and others ; Gehring and others ; Ravindranath and others ; Zhao and others ; Yang and others ; Wang and others The efficiency and selectivity of magnetic separation greatly depends on the ligands, as sometimes it is hard to obtain a ligand with high affinity and specificity to the target.

In addition, the size of MNPs is 1 to 2 orders of magnitude smaller than bacterial pathogens, which allows multiple MNPs to attach to 1 bacterial cell and improve the separation Wang and others Different compounds and procedures have been used to stabilize the magnetic core, improve surface functionalization, and minimize nonspecific interactions Wang and others ; Li and others b ; Cloutier and others The longevity of the biological material attached to magnetic particles, interferences in food matrices, the strength of the magnetic field, and sufficient mixing to maximize the interactions between the functionalized particles and targets need to be considered when using the magnetic separation technique.

In most cases, IMS cannot differentiate dead cells from viable cells, which are the most critical for food contamination. Usually, the pathogens attached to the magnetic beads are not detached during the detection. Therefore, the interference of magnetic beads to detection signals needs to be considered. Instead of functionalized magnetic beads to remove the target pathogens from food samples, immobilized molecules on solid surfaces or materials are used to directly capture the target to the detector surface for analysis, and unbound substances in the samples are removed by washing steps Taylor and others ; Waswa and others ; Chen and others ; Wang and others In addition, since the food matrices are introduced and directly contact with the detection system, such as a chip surface of a biosensor, the washing step is critical and the regeneration of the surface is needed if the chip is not disposable.

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The pretreatment methods applied to bacteria affect the detection. Taylor and others a compared three methods of preparing E. The treatment changed the shape or the size of the bacteria by breaking the bacterial cells into small pieces, which further affected the number, uniformity, and the diffusion of the analytes. Overall, the effect on cell viability should be considered according to the sample requirement of a detection method Stevens and Jaykus Some foodborne pathogens, including C.

Results of these assays are easy to interpret due to the specificity of this type of detection. PCR assays for the detection of foodborne pathogens and toxins in a variety of food matrices have been developed and validated. A study looking at the detection of S. The nanoparticles were incubated with the extracted DNA for 30 min, followed by collection with a magnet.

The particles were able to directly bind to a trace amount of genomic DNA under optimized conditions. PCR using primers to genomic regions specific to both S. Enteritidis and L. PCR was also used to develop a detection method of C. Contaminated food matrices were pretreated with sedimentation and centrifugation techniques to remove coarse particles and solidified fat.

Following DNA extraction, PCR was conducted with primers specific to genomic regions of the pathogen, followed by electrophoretic detection. Also using beef as a matrix, a study looked at the detection of E. Metal hydroxides are charged particles with high molecular weights that are used as affinity agents to bacterial cells Dwivedi and Jaykus Food matrix samples were homogenized and underwent a centrifugation process prior to DNA extraction.

Instead of only 1 set of primers, mPCR uses multiple sets of primers capable of each detecting a gene, a gene variant, or a genomic marker of a specific organism. Therefore, mPCR is advantageous in that less time and effort is required to produce the same detection results. Since implemented in Chamberlain and others , mPCR has been widely used to study foodborne pathogen detection. An mPCR study used milk and chicken meat matrices to test the detection of S. An mPCR assay to simultaneously detect E. Another mPCR assay was developed for the detection of S.

Although a robust and specific assay for detecting foodborne pathogens and toxins in foods, a limitation of the mPCR technique is that the primers used need to be carefully designed with similar annealing temperatures. Since the mPCR assay is a single reaction, all primers will need to anneal during the same temperature to be efficient.

The concentration of each primer also needs to be validated, due to the possibility of interactions between primer sets that may result in primer dimers or amplification of unwanted target sequences Zhao and others Other limitations to the assay include the need for optimizing buffer concentrations, magnesium chloride concentrations, the amount of dNTPs, the amount of template DNA, and the proper cycling temperatures Markoulatos and others Generally, primers should not be much higher than a concentration of 0. The assay offers simultaneous amplification and detection of target DNA.

Amplified DNA is detected using a fluorescently labeled moiety which is detected by the thermocycler at every cycle Dwivedi and Jaykus An increase of fluorescence is compared with the cycle number to generate an amplification curve. From this curve, a quantification cycle Cq or Ct can be determined. The Cq value represents the amount of fluorescence which is significantly higher than the background Postollec and others An increase in DNA was directly proportional to the fluorescence intensity.

The amplified DNA is quantified by measuring the fluorescence intensity of the intercalating dye. For example, a study looking at Salmonella spp. To determine if amplified PCR products are the correct size, a melting curve analysis can be conducted; a single peak indicates one melting temperature and thus one amplicon.

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Louis, Mo. This results in an increased fluorescence which can be read by the thermocycler. Therefore, amplification is quantified based on the fluorescence of the nucleotide which is released from the internal probe. The assay targeted 2 genes specific to the pathogen including the Shiga toxin II gene. Both assays were capable of detecting S. Another assay using PMA pretreatment studied the simultaneous detection of E.

Another advantage of this technique is that the relative or quantitative mRNA levels of an organism under certain conditions can be deduced. This could apply to the food matrix composition, the temperature, the pH or water activity, or the amount of oxygen Zhao and others The method coupled the use of metal hydroxides to immobilize bacterial cells before RNA extraction.

In a LAMP reaction, there are 4 primers, 2 inner and 2 outer, which target 6 to 8 specific sequences. This allows for higher specificity than general PCR Xu and others Like PCR amplicons, LAMP amplicons can be detected by fluorescence in real time or by gel electrophoresis and appropriate staining methods. The assay was compared with previously developed qPCR assays and found to be equally sensitive.

The assay used centrifugation and PMA pretreatment and could detect 6.

The assay could detect 5. The method is used for the detection of specific RNA sequences in vitro. These transcribed copies become the template for another round of reactions as with general PCR, the assay results in exponential amplification of the target product, in this case RNA. This technique has been used for foodborne pathogen detection, including the detection of viable cells Churruca and others The probes on the device are generally short sequences ranging from 25 to 80 bp and are complementary to genes or genomic markers of different target pathogenic organisms Severgnini and others The fluorescence signal intensity is proportional to the concentration of the target DNA sequence Lauri and Mariani Related to foodborne pathogenic bacteria, one of the first studies using microarray technology used the assay for the simultaneous detection of 22 pathogens from broth cultures Wang and others Some of the pathogens that this assay was able to detect were L.

A study using microarray technology developed an assay for the detection of Y. Another assay using milk as a food matrix was developed to detect L. It is noted that the 2 aforementioned studies were able to detect their targets only at very high concentrations. Oligonucleotide microarray technology is generally considered advantageous in that it is highly sensitive and specific to target sequences, it can detect multiple pathogenic organisms at once, and is not labor intensive.

However, the assay requires the production of the oligonucleotide probes and fluorescent labeling of input DNA sequences, which can be expensive. Various commercial kits are available for detection studies; however, there exist few arrays kits specific for food applications Rasooly and Herold Various steps, such as sample preparation, enrichment, labeling, signal amplification, and detection, are all conducted within channels and wells that are etched into the chip.

Since food samples are often complex in nature and may only contain trace amounts of pathogenic organisms of concern, the organisms or associated DNA should be extracted from the food matrix before sample insertion into the LOC Lui and others Usually small volumes are introduced into microfluidic systems; therefore, techniques should be utilized to concentrate the organism or DNA of concern. In some LOCs, organisms can be purified from a heterogeneous sample using methods employing magnetic particles.

This procedure can also be used with food matrices in which target organisms are not homogeneously distributed. The amplified DNA can be visualized by optical methods or by fluorescence signal detection Yoon and Kim Microfluidic LOC devices have been applied to the detection of pathogens such as S. Enteritidis, S. This assay could detect 3. Many biosensors are also based on antibody—antigen interactions, but those techniques involving signal transduction and processing will be discussed in a later section. ELISA is one of the most widely used assays for foodborne pathogen detection.

Typhimurium in spiked food samples within 2 h of inoculation. Combined with MNPs for target separation, they detected E. There have been various reports on the selection and development of antibodies for foodborne pathogens Sunwoo and others ; Charlermroj and others ; Meyer and others Based on the configuration of ELISA, variants for better sensitivity, shorter detection time, and capability of multiplex detection have been developed. Instead of traditional color change indication, fluorescent and chemiluminescent labels are frequently reported. Karoonuthaisiri and others reported a sandwich chemiluminescent immunoassay based on enzyme catalysis for simultaneous detection of E.

Typhimurium, which was evaluated in spiked milk samples. A multiplex sandwich immunoassay combined with IMS and fluorescent detection was reported by Cho and others for the highly sensitive detection of E. Typhimurium within 2 h. The detection was achieved by enzymatic digestion to release fluorophores which attached to the magnetically captured target.

Lateral flow along the membrane strip provides a simple and rapid form of detection assay. Usually, a test result is indicated by color change, which is provided by AuNPs or enzymatic reactions Shim and others ; Park and others A strip test using AuNPs as indicators for the detection of V. Recent developments in lateral flow assays include the development of novel labels, new formats of assays, new supporting materials, and multiplex detection Shan and others Some assays combine the test strip with devices to measure the color intensities or biosensor technology to be correlated with analyte concentration.

Hossain and others reported a test strip on paper for multiplex detection of E. Two enzymes and their substrates were used to indicate the presence of the 2 bacteria, respectively, and a hydrophobic barrier was used to prevent the interference between the 2 detection zones. This assay was also combined with IMS to improve sensitivity and was used for quantitative detection with the measurement of color intensity.

Chai and others developed a portable detection system to detect the reflecting light from the reacting area on a test strip. Compared to conventional culture methods, biosensors have the advantages of less detection time and relatively simple manipulation which does not require highly trained personnel.

Biosensors can achieve the similar, or even better, sensitivity than other detection methods. In this section, biosensors without cultural enrichment after inoculation are reviewed, especially those which have seen development in recent years. The biosensors are categorized based on their transducing mechanisms.

Key performance characteristics such as detection time, LOD, detection range, and specificity are summarized in Table 3. Optical biosensors are the most widely reported biosensors in foodborne pathogen detection, and there are now commercialized optical biosensors available.

Nanomaterials such as nanofibers, nanoparticles, and quantum dots have attracted much interest due to their unique optical properties, and their applications in biosensors have been reported Leung and others ; Vinayaka and Thakur Because SPR sensors monitor the interactions in very close vicinity of the transducer surface, it has the potential to study the interactions without rinsing out unreacted or excess reactants Shankaran and others Wei and others reported a SPR biosensor for the detection of C. Antibody against C. It is noted that there was significant interference from the matrices, such as the proteins and lipid compounds.

Waswa and others described an SPR immunosensor for E.

Bacterial Pathogenesis

A sandwich assay was employed for the detection of E. Antibodies against C. Miniaturized SPR biosensor, multiplex detection, and nanomaterials have attracted much interest in recent years. Biacore launched several SPR sensors which comprised multiple flow cells enabling serial or parallel injections of samples in Situ and others Typhimurium, L. Instead of using antibodies, other sensing elements have been explored for SPR biosensors. For example, lectin wheat germ agglutinin from Triticum vulgaris was used as a bioreceptor for the detection of E.

The LOD of E. The coupling of SPR and other technologies also led to diverse possibilities to improve the detection performance, such as SPR coupled with mass spectrometry Zhukov and others ; Chen and others ; Nedelkov , and SPR combined with fluorescence imaging Zordan and others Although these systems have not been evaluated for the detection of pathogens in food, they can potentially be used for this purpose. Fluorescence resonance energy transfer FRET is a phenomenon of nonradiative energy transfer from a fluorescent donor molecule to an acceptor molecule due to dipole—dipole interactions when in close proximity Lakowicz This biosensor was used to detect S.

Other optical methods have been implemented for biosensors for foodborne pathogen detection in food matrices without enrichment. Functionalized polymer vesicles, which respond to bacterial metabolites, were synthesized and used for colorimetric detection of S. The color response was analyzed using a colorimeter, and a spectrometer was used to obtain spectra of the vesicles.

You and others developed a handheld device based on light scattering in latex immunoagglutination assays. Filtered lettuce solution spiked with E. Enteritidis 16S rRNA. An electrochemical measurement can be applied to liquid, solid, or gaseous analytes, but the latter 2 are not common for food sample analysis. Electrochemical biosensors have good sensitivity, fast response, and simplicity Sharma and Mutharasan a. Compared to SPR in a study for E.

The electrochemical biosensors have been the most commonly used and have received the most interest during the past 2 decades, and many studies have successfully detected foodborne bacterial pathogens in food samples. Viswanathan and others reported a disposable voltammetric sandwich immunosensor for multiplex detection of E. Milk spiked with the pathogens was used as the food matrix for evaluation. Selective membranes are often used to minimize the interferences Wahono and others Detection strategies involving the changes in pH or ion concentration can be coupled with potentiometric measurements which are able to detect ions in a wide concentration range Palchetti and Mascini A filtration system was used to remove the charged species in food samples.

There have not been many applications of these biosensors for bacterial pathogen detection during the last decade, mainly due to the incompatibility of biomolecule immobilization methods with ISFET fabrication and inadequate device stability Palchetti and Mascini ; Sharma and Mutharasan a.

Rapid single-colony whole-genome sequencing of bacterial pathogens.

Pal and others developed a sandwich conductometric immunosensor on membrane pads to detect B. The liquid sample was moved through the pad by lateral flow, and the changes in resistance due to the formation of the sandwich structure involving polyaniline when the target presented was measured. Changes in the resistance were measured, and the biosensor was evaluated with spiked milk and baby food. Impedimetric biosensors are commonly run using an electrochemical impedance spectroscopy technique, which analyzes both the resistive and capacitive properties of biological components.

Listeria innocua , which is a surrogate for L. Joung and others used an alumina nanoporous membrane with hyaluronic acid as the transducer and immobilized antibody on the membrane for E. Many different configurations of impedimetric biosensors are available due to the advantages of the measurement methods, and they are expected to continue attracting research interest as portable and rapid detection devices. Piezoelectric quartz crystal microbalance QCM is one major type of piezoelectric biosensor which has been investigated for pathogen detection.

They reported a detection limit of 0. Nanogram levels of mass changes can be detected by QCM sensors, which is usually not low enough for sensitive detection. Therefore, mass amplification is a commonly used approach for improving the sensitivity. New sensing elements and effective ligand immobilization approaches are also under development. The polymeric structure for target recognition was developed via contact imprinting of whole cells. Although the detection limit is not satisfactory, the studies using molecular imprinting polymers indicate a potential new detection strategy.

Cantilever sensors based on piezoelectric materials for mass sensing have also been reported for pathogen detection. The PEMC sensor comprises a piezoelectric layer which provides a sensitive response to weak stresses. Magnetoelastic ME sensors are made of amorphous ferromagnetic alloys.


Several studies reported the detection of foodborne pathogens on the surface of fresh produce with minimal or no sample preparation. Filamentous E2 phages were immobilized on ME sensors and the sensors were placed on tomatoes for 30 min to bind S. Typhimurium on the surface. The resonance frequency, which changes before and after contact with tomatoes, was measured Li and others ME biosensors also showed comparable sensitivity and repeatability as qPCR but required less time and skill to manipulate, for detecting S.

Typhimurium on spinach leaves and cantaloupes Park and others a , b. For these ME biosensor studies, S. Nanotechnology has been introduced in biosensors due to the special properties which facilitate detection. Developments in new transducing materials, new recognition approaches, multiplex detection configurations, microfluidic techniques, integrated biosensing systems for example, LOC , and wireless devices would promote biosensors to become mature products for direct application in the food industry.

Since foodborne pathogens are generally present in food samples at low concentrations, novel preprocessing and rapid detection methodologies are required to work with these low numbers. Many methods to detect foodborne pathogens have been developed in recent years to address the attention of food safety and public health concerns, especially with the increasing demands of fresh and minimally processed food products.

However, the sensitivity of the assays is one of the major concerns of using these methods. Many studies have improved the sensitivity and enabled multiplex detection. Automation of these assays is also under investigation. Different sensing elements, transducing technologies and configurations of biosensors have been reported in recent years for direct detection of foodborne pathogens. Effective and rapid separation methods, highly specific detection probes, and comprehensive validation of new assays will aid in the development of future detection technologies.

The authors gratefully thank Mary Lou Tortorello for helpful discussions while preparing the manuscript. Food and Drug Administration. The sponsors had no role in the study design, data collection, and analysis, decision to publish, or preparation of the manuscript. YW provided the idea of the manuscript. YW and JKS developed and wrote the manuscript. Both authors read, edited, and approved the final manuscript.

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