Tool For Detection Of Pathogens Research Articles

Diagnostic efficacy of metagenomic next-generation sequencing for intracranial bacterial infection pathogens after neurosurgery

Objective: To explore the diagnostic efficacy of metagenomicnext-generation sequencing (mNGS) technique for pathogen diagnosis of intracranial infection after neurosurgery. Methods: Patients with suspected intracranial infection after neurosurgery who were treated in Beijing Tiantan Hospital of Capital Medical University from May 2017 to October 2018 were selected. Cerebrospinal fluid samples were collected for mNGS detection and bacterial culture. The sensitivity, specificity, positive predictive value and negative predictive value of these two methods were calculated, and their differences were compared. Results: A total of 80 cerebrospinal fluid samples from patient with suspected intracranial infection after neurosurgery were included, including 53 males and 27 females, with a mean age of (41±19) years old(age range: 2-80 years).After clinical review, a clinical diagnosis was made by two neurosurgery specialists through comprehensively interpretation of the patient's clinical data, laboratory tests and imaging examinations. Finally, 42 cases of intracranial infection and 38 cases of non-infection were clinically diagnosed. The sensitivity and specificity of mNGS detection were 83.33%(35/42) and 76.32%(29/38), and the positive predictive value and negative predictive value were 79.55%(35/44) and 80.56%(29/36). Meanwhile, the sensitivity and specificity of bacterial culture were 59.52%(28/42) and 68.42%(26/38), the positive predictive value and negative predictive value were 68.00% (28/40) and 60.47%(26/40). The sensitivity of mNGS detection washigher than that of bacterial culture, and the difference was statistically significant(χ2=5.83, P=0.015).Compared with bacterial culture, there was no statistically significant difference in the specificity of mNGS detection(χ2=0.59, P=0.441). Conclusion: mNGS detection technique can improve the detection rate of intracranial infection pathogens after neurosurgery, and may become a promising auxiliary diagnostic tool for pathogen detection.

Update and Validation of the 16S rDNA qPCR Assay for the Detection of Three ‘Candidatus Liberibacter Species’ Following Current MIQE Guidelines and Workflow

An updated real-time multiplex quantitative polymerase chain reaction (qPCR) assay was designed and validated for the simultaneous detection of three ‘ Candidatus Liberibacter species’ (CLsp), ‘ Ca. Liberibacter asiaticus’ (CLas), ‘africanus’ (CLaf), and ‘americanus’ (CLam), associated with the huanglongbing disease of citrus. The multiplex assay was designed based on the qPCR assay published in 2006 by Li et al., considering all available CLsp 16S rRNA gene sequences in GenBank and the MIQE guidelines and workflow for qPCR optimization, which became available after 2006. When using the updated multiplex CLsp qPCR assay compared with singleplex qPCR, no significant increase in quantitative cycle (Cq) values was detected. The specificity and sensitivity of the updated qPCR assay was optimal, and measuring the intra- and interassay variations confirmed the reproducibility and repeatability of the assay. The assay was also successfully used with a large number of diverse samples at independent laboratories in four countries, thus demonstrating its transferability, applicability, practicability, and robustness as different qPCR reaction conditions or instruments had a minor effect on Cq values. This updated multiplex CLsp qPCR assay can be used in a variety of citrus surveys, germplasm, or nursery stock programs that require different pathogen detection tools for their successful operation. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Recent Advances in CRISPR-Based Biosensors for Point-of-Care Pathogen Detection.

Infectious pathogens are pressing concerns due to their heavy toll on global health and socioeconomic infrastructure. Rapid, sensitive, and specific pathogen detection methods are needed more than ever to control disease spreading. The fast evolution of clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics (CRISPR-Dx) has opened a new horizon in the field of molecular diagnostics. This review highlights recent efforts in configuring CRISPR technology as an efficient diagnostic tool for pathogen detection. It starts with a brief introduction of different CRISPR-Cas effectors and their working principles for disease diagnosis. It then focuses on the evolution of laboratory-based CRISPR technology toward a potential point-of-care test, including the development of new signaling mechanisms, elimination of preamplification and sample pretreatment steps, and miniaturization of CRISPR reactions on digital assay chips and lateral flow devices. In addition, promising examples of CRISPR-Dx for pathogen detection in various real samples, such as blood, saliva, nasal swab, plant, and food samples, are highlighted. Finally, the challenges and perspectives of future development of CRISPR-Dx for infectious disease monitoring are discussed.

Nucleic Acids Detection for Mycobacterium tuberculosis Based on Gold Nanoparticles Counting and Rolling-Circle Amplification.

Tuberculosis (TB) is a common infectious disease caused by Mycobacterium tuberculosis, which usually disturbs the lungs, and remains the second leading cause of death from an infectious disease worldwide after the human immunodeficiency virus. Herein, we constructed a simple and sensitive method for Mycobacterium tuberculosis-specific DNA detection with the dark-field microscopic imaging of gold nanoparticles (AuNPs) counting strategy and rolling-circle amplification (RCA). Taking advantage of RCA amplification, one target molecule produced hundreds of general oligonucleotides, which could form the sandwich structure with capture-strand-modified magnetic beads and AuNPs. After magnetic separation, AuNPs were released and detected by dark-field imaging; about 10 fM Mycobacterium tuberculosis-specific DNA target can still be differentiated from the blank. No significant change of the absorbance signals was observed when the target DNA to genomic DNA ratio (in mass) was from 1:0 to 1:106. The spike recovery results in genomic DNA from human and Klebsiella pneumoniae suggested that the proposed method has the feasibility for application with biological samples. This proposed method is performed on an entry-level dark-field microscope setup with only a 6 μL detection volume, which creates a new, simple, sensitive, and valuable tool for pathogen detection.

Keeping up with advances in qPCR pathogen detection: an example for QPX disease in hard clams.

With marine diseases on the rise and increased reliance on molecular tools for disease surveillance, validated pathogen detection capabilities are important for effective management, mitigation, and response to disease outbreaks. At the same time, in an era of continual evolution and advancement of molecular tools for pathogen detection, it is critical to regularly reassess previously established assays to incorporate improvements of common practices and procedures, such as the minimum information for publication of quantitative real-time PCR experiments (MIQE) guidelines. Here, we reassessed, re-optimized, and improved the quantitative PCR (qPCR) assay routinely used for Quahog Parasite Unknown (QPX) disease monitoring. We made 19 significant changes to the qPCR assay, including improvements to PCR amplification efficiency, DNA extraction efficiency, inhibition testing, incorporation of linearized standards for absolute quantification, an inter-plate calibration technique, and improved conversion from copy number to number of cells. These changes made the assay a more effective and efficient tool for disease monitoring and pathogen detection, with an improved linear relationship with histopathology compared to the previous version of the assay. To support the wide adoption of validated qPCR assays for marine pathogens, we provide a simple workflow that can be applied to the development of new assays, re-optimization of old or suboptimal assays, or assay validation after changes to the protocol and a MIQE-compliant checklist that should accompany any published qPCR diagnostic assay to increase experimental transparency and reproducibility amongst laboratories.

Enhanced electrochemiluminescence bioassay triggered by intracellular leakage for detection of Escherichia coli

An intracellular leakage-trigged signal-on solid-state electrochemiluminescent (ECL) assay is developed for the detection of Escherichia coli (E. coli). A self-assembled multilayer ensemble of N, S co-doped carbon dots -poly dimethyl diallylammonium chloride grafted carbon nanospheres is used as ECL luminophores with peroxydisulfate (PS) ions as coreactants. The incorporation of molecularly imprinted electrospun nanofibers with the multilayer ensemble enables a robust, highly selective solid-state ECL probe without using any expensive and fragile biological receptor. Upon the imprinted E. coli exposed to the assay, under bactericidal effects of PS ions by destroying the integrity of E. coli cell membrane, intracellular leakage K+-triggered ECL enhancement is first disclosed via prompting the involved 1O2-mediated ECL process. Benefiting from the ECL enhancement upon increasing the concentration of E. coli, a unique intracellular leakage-trigged signal-on ECL system is created for sensing E. coli. Such a assay is proved to be highly specific and sensitive for sensing E. coli in the concentration range from 5 to 107 cfu mL−1, achieving a detection limit of 1 cfu mL−1 (S/N = 3). This label-free, simple and facile assay provides a promising point-of-care diagnostic tool for pathogen detection.

SaQuant: a real-time PCR assay for quantitative assessment of Staphylococcus aureus

BackgroundMolecular assays are important tools for pathogen detection but need to be periodically re-evaluated with the discovery of additional genetic diversity that may cause assays to exclude target taxa or include non-target taxa. A single well-developed assay can find broad application across research, clinical, and industrial settings. Pathogen prevalence within a population is estimated using such assays and accurate results are critical for formulating effective public health policies and guiding future research. A variety of assays for the detection of Staphylococcus aureus are currently available. The utility of commercial assays for research is limited, given proprietary signatures and lack of transparent validation.ResultsIn silico testing of existing peer-reviewed assays show that most suffer from a lack of sensitivity and specificity. We found no assays that were specifically designed and validated for quantitative use. Here we present a qPCR assay, SaQuant, for the detection and quantification of S. aureus as might be collected on sampling swabs. Sensitivity and specificity of the assay was 95.6 and 99.9 %, respectively, with a limit of detection of between 3 and 5 genome equivalents and a limit of quantification of 8.27 genome equivalents. The presence of DNA from non-target species likely to be found in a swab sample, did not impact qualitative or quantitative abilities of the assay.ConclusionsThis assay has the potential to serve as a valuable tool for the accurate detection and quantification of S. aureus collected from human body sites in order to better understand the dynamics of prevalence and transmission in community settings.

Study on bacterial pathogens through multiplex polymerase chain reaction system and their antimicrobial resistance pattern in goats presumed with fever and/or diarrhea.

Background and Aim:Goat is one of the major livestock species, plays an important role in the economy of Bangladesh. However, the outbreak of different infectious diseases in goats causes high mortality and economic losses due to lack of proper diagnosis and treatment. Conventional culture-based methods for detecting specific pathogens as confirmatory diagnosis are laborious as well as time-consuming in comparison to multiplex polymerase chain reaction (mPCR), by which multiple pathogens can be detected at a time. The present study was aimed to perform faster molecular identification of bacterial pathogens from goats presumed with fever and/or diarrhea and their antimicrobial resistance (AMR) pattern.Materials and Methods:A total of 200 blood samples were collected from goats at S. A. Quaderi Teaching Veterinary Hospital (SAQTVH) in Chattogram Veterinary and Animal Sciences University for the period of July 2017-April 2018. DNA was extracted and subsequently, mPCR assay was performed for the screening of several bacterial pathogens (Salmonella spp., Listeria monocytogenes, Bacillus cereus, Yersinia enterocolitica, Campylobacter jejuni, Campylobacter coli, Clostridium perfringens, Vibrio cholerae, and Staphylococcus aureus). An antimicrobial susceptibility test against ten antimicrobials for positive samples of each organism was conducted using the Kirby–Bauer Disk-Diffusion Method on selective media.Results:S. aureus, C. perfringens, L. monocytogenes, and Salmonella spp. were detected from collected samples and their overall prevalence was 11.5%, 3.5%, 1%, and 20.5%, respectively. The most common clinical signs were mild fever, nasal discharge, dyspnea, and coughing (39.1%) for S. aureus, diarrhea, convulsion, abdominal pain, and incoordination (57.1%) for C. perfringens, fever, protrusion of tongue, and incoordination (100%) for L. monocytogenes, and fever, anorexia, dehydration with mucous feces (36.6%) for Salmonella spp. infection in goats. AntimGentamicinicrobial diagram of S. aureus showed resistance against Cefotaxime (74%), Cefixime (65%), and Tetracycline (65%); highly sensitive against Amoxicillin (48%), Ciprofloxacin (44%), and Gentamicin (44%). On the other hand, C. perfringens showed highly resistant against Ampicillin (71%), Gentamicin (71%), sensitive against Penicillin (57%), and Cefotaxime (57%). L. monocytogenes were found to be sensitive to Penicillin (100%) and Cefixime (100%) and Salmonella spp. showed resistance to Ampicillin (78%) and Amoxicillin (59%) but sensitive to Ciprofloxacin (54%).Conclusion:This study identified pathogens with their specific clinical signs in goats presumed fever and/or diarrhea through mPCR with their AMR pattern in SAQTVH, Chattogram. Potential risk factors, measuring the strength of association of disease caused by these particular pathogens, were also determined. mPCR may use as an effective tool for rapid detection of pathogens in animal.

MegaPath: sensitive and rapid pathogen detection using metagenomic NGS data

BackgroundNext-generation sequencing (NGS) enables unbiased detection of pathogens by mapping the sequencing reads of a patient sample to the known reference sequence of bacteria and viruses. However, for a new pathogen without a reference sequence of a close relative, or with a high load of mutations compared to its predecessors, read mapping fails due to a low similarity between the pathogen and reference sequence, which in turn leads to insensitive and inaccurate pathogen detection outcomes.ResultsWe developed MegaPath, which runs fast and provides high sensitivity in detecting new pathogens. In MegaPath, we have implemented and tested a combination of polishing techniques to remove non-informative human reads and spurious alignments. MegaPath applies a global optimization to the read alignments and reassigns the reads incorrectly aligned to multiple species to a unique species. The reassignment not only significantly increased the number of reads aligned to distant pathogens, but also significantly reduced incorrect alignments. MegaPath implements an enhanced maximum-exact-match prefix seeding strategy and a SIMD-accelerated Smith-Waterman algorithm to run fast.ConclusionsIn our benchmarks, MegaPath demonstrated superior sensitivity by detecting eight times more reads from a low-similarity pathogen than other tools. Meanwhile, MegaPath ran much faster than the other state-of-the-art alignment-based pathogen detection tools (and compariable with the less sensitivity profile-based pathogen detection tools). The running time of MegaPath is about 20 min on a typical 1 Gb dataset.

Nanoporous layer fiber biosensing platform for real time culture- and separation-free detecting bacterial pathogens and measuring their susceptibility to antibiotics

Simple, rapid, and sensitive detection of bacterial pathogens in environmental and clinical samples is a key requirement because of the ensuing high morbidity and mortality. In this study, a nanoporous layer fiber biosensing platform (called NFB) was developed for the rapid culture- and separation-free detection of bacterial pathogens through integrating evanescent wave fluorescence technology, the size effect of nanoporous layer, and real-time aptasensors. The in-situ etched nanoporous layer of the fiber probe can not only effectively distinguish free Cy5.5-aptasensors and Cy5.5-aptasensors bound to pathogens but also enhance evanescent wave fluorescence. NFB can selectively and sensitively quantify Escherichia coli O157:H7 and Salmonella typhimurium species in environmental and blood samples within 18 min and 32 min, respectively. NFB detected a significant increase in fluorescence when the mixture of pathogens and Cy5.5-aptasensors was exposed to antibiotics. Our results clearly demonstrated that the portable NFB can serve as a powerful tool for real time detection of pathogens and measurement of their response to antibiotics.

Wild Wild West: Emerging Viruses and Viroids of Hemp

Hemp (Cannabis sativa) or industrial hemp is a rapidly growing industry in the United States. However, the crop has been poorly studied in the US since its production declined in the late 1950s, and information on the biological and agricultural factors affecting hemp is limited. With the passage of the 2014 Farm Bill, hemp is no longer considered an illegal plant and is now a legal agricultural crop in the US. Within the bill, Section 7606 (Legitimacy of Industrial Hemp Research) provided a formal definition of the crop as "the plant Cannabis sativa L. and any part of such plant, whether growing or not, with a delta-9-tetrahydrocannabinol (THC) concentration of not more than 0.3 percent on a dry weight basis". In addition, the 2018 Farm Bill addressed many issues that had previously impeded development of the crop. Currently, 46 US states have enacted laws removing barriers to its production. In 2019, hemp production was 206, 965 hectares with 16,877 grower licenses across 34 states, according to "US Hemp Report" (www.votehemp.com). This is a greater than 455% increase over 2018 licensed acreage. Colorado, the leading state in hemp production increased production from 4,873 licensed hectares (346 growers) in 2017 to 12,525 licensed hectares (835 growers) in 2018. The US retail sales of hemp products was reported at $1.2 billion in 2018. The most profitable market for North American hemp is oilseed production and cannabidiol (CBD), a nonintoxicant cannabinoid with promising therapeutic use as a pharmaceutical product. Current CBD sales are estimated at $190 million and are projected to reach $2.5 billion by 2022. As we enter this new era of hemp production, plant diseases associated with the crop in the US and associated disease management needs are essentially undescribed. The emergence of phytopathogens and their spread is a rising concern as hemp production increases. Therefore, there is an urgent need to identify emerging and established hemp viruses and viroids, elucidate their diversity and distribution, develop early pathogen detection tools, and provide hemp growers, industry and other stakeholders information and resources to make timely management decision and minimize crop loss.

A treponemal genome from an historic plague victim supports a recent emergence of yaws and its presence in 15th century Europe

Developments in techniques for identification of pathogen DNA in archaeological samples can expand our resolution of disease detection. Our application of a non-targeted molecular screening tool for the parallel detection of pathogens in historical plague victims from post-medieval Lithuania revealed the presence of more than one active disease in one individual. In addition to Yersinia pestis, we detected and genomically characterized a septic infection of Treponema pallidum pertenue, a subtype of the treponemal disease family recognised as the cause of the tropical disease yaws. Our finding in northern Europe of a disease that is currently restricted to equatorial regions is interpreted within an historical framework of intercontinental trade and potential disease movements. Through this we offer an alternative hypothesis for the history and evolution of the treponemal diseases, and posit that yaws be considered an important contributor to the sudden epidemic of late 15th century Europe that is widely ascribed to syphilis.

Electrochemical Detection of Endotoxins from Gram-Negative Bacteria Using Immobilized TLR4 Immunoproteins

Diagnostic platforms utilizing biorecognition elements, such as antibodies and DNA molecules, have been demonstrated as useful tools for the rapid detection of pathogens. However, these bioelements are extremely specific to a particular microorganism, which for the purpose of detection, requires prior knowledge of the infection to utilize the proper receptor. In order to circumvent this challenge, our group has been developing biosensors using a family of immunoproteins belonging to the non-specific innate immune system, the Toll-Like Receptor (TLR) immunoproteins, which are programmed to recognize chemical markers from a broad source of microorganisms. This makes them ideal for the first line of detection against an unknown pathogen. For example, TLR4 proteins are selective against endotoxins found in the cell walls of Gram-negative bacteria but not in Gram-positive bacteria. Here we show the results obtained from a Gram-negative bacteria sensor based on TLR4 tethered on the surface of a self-assembled monolayer (SAM) of alkyl thiols on a Au electrode.The TLR4 biosensor assembly involves a multi-step process, starting with the co-adsorption of three alkanethiols onto Au. The resulting SAM possesses distinct characteristics derived from each alkanethiol molecule. A hydroxyl-terminated alkanethiol serves as an anti-fouling agent and a spacer, a ferrocene- (Fc-) terminated alkanethiol is the redox active molecule assisting in charge transfer mediation, and an alkanethiol with a nitrilotriacetic acid- (NTA-) motif permits the chelation of Ni2+ cations, which then immobilizes the TLR4 immunoprotein via its His-tagged recombined C-terminus. Additionally, the desired surface orientation of the TLR4 ligand-recognition sites is also achieved using this anchoring method.1 In nature, TLR4 is a transmembrane monomer that undergoes dimerization once bound to lipopolysaccharide (LPS) endotoxin.2 It is believed that the recombinant TLR4 in our sensors behaves similarly and that dimerization results in an increase in charge transfer resistance (RCT ) with the addition of endotoxin.The sensor assembly process was monitored using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques. CVs were recorded in 200 mM phosphate buffer (PB) pH 7 while tracking the Fc redox chemistry. Similarly, EIS was performed in an electrolyte containing 5 mM ferrocyanide (ferroCN) dissolved in the same buffer. The addition of ferroCN as a second redox probe allows us to monitor the charge transfer mediation between the SAM-bound Fc and the solution-phase ferroCN ions.3 During sensor assembly, CVs showed a peak shift to higher potentials, while EIS showed higher RCT values with the transition from unmodified SAM to TLR4 modification. Both techniques indicated successful assembly. Overall, the sensors show a stable RCT signal prior to LPS exposure. During sensor testing, the amount of LPS added to the sensor was monitored using EIS, tracking the RCT value, as described above. The addition of LPS resulted in a linear RCT change with logarithmic changes in the LPS concentration in the range of 0.01-100 ug/mL, while at higher concentrations, the response deviated from linearity. Early results estimated a limit of detection of 1 ug/mL LPS.Finally, the TLR4-endotoxin interaction has been characterized using the fluorescence-based microscale thermophoresis (MST) technique. In an MST experiment, a fluorophore-labeled molecule is subjected to a pulse of IR laser to heat a focused point in a solution inside a glass capillary, thus inducing microscopic temperature gradients and thermophoresis-induced migration away from that point.4 In our MST experiments, LPS concentrations from 1-2000 ug/mL were combined with a fixed 20 nM fluorescently-labelled TLR4 protein in order to determine their binding affinity. The MST binding curves of TLR4 with endotoxin allowed the determination of the dissociation constant, Kd, which was found to be ca. 11 uM.

New Molecular Tool for a Quick and Easy Detection of Apple Scab in the Field

Venturia inaequalis, an agent of apple scab, is the most important pathogen of Malus x domestica. Control measures against this pathogen rely on intensive phytosanitary programs based on predictive models to identify the meteorological conditions conducive to the primary infection. The detection of the pathogen in field, both in naturally infected symptomatic and asymptomatic leaves, is desirable. Loop-mediated isothermal amplification (LAMP) assays are profitable molecular diagnostic tools for the direct detection of pathogens in field. A LAMP assay for V. inaequalis has been designed on the elongation factor 1-alpha sequence. The validation of the LAMP assay was carried out following the international EPPO standard PM 7/98 in terms of specificity, sensitivity, repeatability and reproducibility. Specificity testing was performed using target and non-target species, such as phylogenetically related Venturia species and other pathogens commonly found in apple, resulting in positive amplification only for the target with a time to positive ranging from 20 to 30 min. Sensitivity testing was performed with serial dilutions of DNA of the target and by artificial inoculation of young apple leaves. The reliability of the LAMP assay as an early-detection tool and its user-friendly application make it suitable for the diagnosis of apple scab in the field.

Development and validation of a multiplex-PCR based assay for the detection of 18 pathogens in the cerebrospinal fluid of hospitalized children with viral encephalitis

BackgroundThe Applied Biosystems 3500 Genetic Analyzer (ABI3500) allows for automated capillary electrophoresis on multiple targets. So far, the application of this method for detecting cerebrospinal fluid pathogens has hardly been reported. MethodsTo assess the performance of multiplex-PCR assay for 18 pathogens detection, 127 CSF samples from hospitalized children with suspected viral encephalitis were prospectively collected from April to November 2018. The Sanger sequencing was applied to verify this assay. ResultsAll of the 18 target pathogens can be identified by multiplex-PCR assay at 104 copies (or CFU/mL) of each virus, bacterium and fungus. In contrast, 10 control microorganisms failed to be amplified. Approximately 68.5 % of the cases tested had positive results, the enterovirus accounted for the majority of the positive cases (63.8 %). Agreement between multiplex-PCR and sequencing was 91.49 %. ConclusionsOur findings suggest that the ABI3500-based multiplex-PCR detection kit could be a valuable diagnostic tool for pathogen detection in CSF of children with suspected viral encephalitis.

Detection of Ralstonia solanacearum phylotype II, race 2 causing Moko disease and validation of genetic resistance observed in the hybrid plantain FHIA-21

Vascular wilt of banana and plantains, also known as Moko disease, is caused by Ralstonia solanacearum (Rs) phylotype II, and is the main bacterial disease affecting these crops in the Americas. Upon comparative sequence analysis of 44 Rs genomes we developed an improved PCR protocol based on the nucleotide sequence of a gene coding for a hypothetical DUF3313 domain protein. Next, we tested the detection protocol with two Rs inoculation methods to validate field resistance reported in the hybrid plantain genotype FHIA-21, previously identified as susceptible to Moko disease in greenhouse experiments in which wounds were caused to the roots prior to inoculation. By using an inoculation method without causing wounds to the roots, we confirmed resistance of FHIA-21 to Moko disease (no Rs was detected by PCR in inoculated plants). In contrast, the field-susceptible genotype Dominico Harton developed severe symptoms of Moko disease, regardless of the inoculation method used. FHIA-21 showed an area under the disease progress curve (AUDPC) close to zero, while Dominico Harton plants showed AUDPC values ranging from 65.8 to 88.4. The availability and analysis of genomic data facilitates the development of improved pathogen detection tools that together with the availability of improved inoculation methods and tolerant genotypes to Moko disease will be of great use in Musa breeding programs.

Engineering Bacteriophages as Versatile Biologics.

Viruses of bacteria (bacteriophages or phages) are highly evolved nanomachines that recognize bacterial cell walls, deliver genetic information, and kill or transform their targets with unparalleled specificity. For a long time, the use of genetically modified phages was limited to phage display approaches and fundamental research. This is mostly because phage engineering has been a complex and time-consuming task, applicable for only a few well characterized model phages. Recent advances in sequencing technology and molecular biology gave rise to rapid and precise tools that enable modification of less-well-characterized phages. These methods will pave the way for the development of modular designer-phages as versatile biologics that efficiently control multidrug-resistant bacteria and provide novel tools for pathogen detection, drug development, and beyond.

Rapid pathogen detection tools in seafood safety

Detection, identification, and monitoring of pathogens in seafood are critical in achieving seafood safety goals. Rapid detection techniques enable outcome of fast and reliable results especially with expansion in the global trade requiring rapid transport over long distances. This review summarizes some of the major techniques in use and recent advances for rapid detection of pathogens in seafood which can be broadly classified into nucleic acid-based, antigen-antibody based, biosensor-based and phage-based methods. Selection of an appropriate pathogen detection method along with a combination of several other state-of-art technologies can as well ensure the effectiveness of pathogen detection and aid in ameliorating seafood safety.

Immobilization of Bacteriocins from Lactic Acid Bacteria and Possibilities for Application in Food Biopreservation

Bacteriocins are biologically active compounds produced by a large number of bacteria, including lactic acid bacteria (LAB), which exhibit antimicrobial activity against various saprophytic and pathogenic microorganisms. In recent decades, bacteriocins are increasingly becoming more important in different branches of the industry due to their broad antibacterial and antifungal spectrum - in the food industry for natural food preservation and expiry date extension; in the health sector for preparation of probiotic foods and beverages; in the clinical practice as alternatives of conventional antibiotics; in the agriculture as biocontrol agents of plant pathogens and alternatives of chemical pesticides for plant protection. The broad antimicrobial spectrum of bacteriocins has stimulated the research attention on their application mainly in the food industry as natural preservatives. Most scientific achievements concerning the application food biopreservation are related to bacteriocins produced by LAB. The lactic acid bacteria bacteriocins can be produced in the food substrate during its natural fermentation or can be added in the food products after obtaining byin vitrofermentations under optimal physical and chemical conditions. Moreover, the immobilization of LAB bacteriocins on different matrices of organic and inorganic origin has been proposed as an advanced approach in the natural food preservation for their specific antimicrobial activity, anti-biofilm properties and potential use as tools for pathogen detection.

Detection and Characterisation of Anaplasma marginale and A. centrale in South Africa.

Bovine anaplasmosis is endemic in South Africa and it has a negative economic impact on cattle farming. An improved understanding of Anaplasma marginale and Anaplasma marginale variety centrale (A. centrale) transmission, together with improved tools for pathogen detection and characterisation, are required to inform best management practices. Direct detection methods currently in use for A. marginale and A. centrale in South Africa are light microscopic examination of tissue and organ smears, conventional, nested, and quantitative real-time polymerase chain reaction (qPCR) assays, and a reverse line blot hybridisation assay. Of these, qPCR is the most sensitive for detection of A. marginale and A. centrale in South Africa. Serological assays also feature in routine diagnostics, but cross-reactions prevent accurate species identification. Recently, genetic characterisation has confirmed that A. marginale and A. centrale are separate species. Diversity studies targeting Msp1a repeats for A. marginale and Msp1aS repeats for A. centrale have revealed high genetic variation and point to correspondingly high levels of variation in A. marginale outer membrane proteins (OMPs), which have been shown to be potential vaccine candidates in North American studies. Information on these OMPs is lacking for South African A. marginale strains and should be considered in future recombinant vaccine development studies, ultimately informing the development of regional or global vaccines.