Multiplexed Bead-Based Mesofluidic System for Detection of Food-Borne Pathogenic Bacteria

ABSTRACT In the present study, a simple and rapid multiplexed bead-based mesofluidic system (BMS) was developed for simultaneous detection of food-borne pathogenic bacteria, including Staphylococcus aureus, Vibrio parahaemolyticus, Listeria monocytogenes, Salmonella, Enterobacter sakazakii, Shigella, Escherichia coli O157:H7, and Campylobacter jejuni. This system is based on utilization of isothiocyanate-modified microbeads that are 250 μm in diameter, which were immobilized with specific amino-modified oligonucleotide probes and placed in polydimethylsiloxane microchannels. PCR products from the pathogens studied were pumped into microchannels to hybridize with the oligonucleotide-modified beads, and hybridization signals were detected using a conventional microarray scanner. The short sequences of nucleic acids (21 bases) and PCR products characteristic of bacterial pathogens could be detected at concentrations of 1 pM and 10 nM, respectively. The detection procedure could be performed in less than 30 min with high sensitivity and specificity. The assay was simple and fast, and the limits of quantification were in the range from 500 to 6,000 CFU/ml for the bacterial species studied. The feasibility of identification of food-borne bacteria was investigated with samples contaminated with bacteria, including milk, egg, and meat samples. The results demonstrated that the BMS method can be used for effective detection of multiple pathogens in different foodstuffs.

Download Full-text

Microbial contamination of raw meat and its environment in retail shops in Karachi, Pakistan

The Journal of Infection in Developing Countries ◽

10.3855/jidc.599 ◽

2010 ◽

Vol 4 (06) ◽

pp. 382-388 ◽

Cited By ~ 30

Author(s):

Nafisa Hassan Ali ◽

Amber Farooqui ◽

Adnan Khan ◽

Ameera Yahya Khan ◽

Shahana Urooj Kazmi

Keyword(s):

Staphylococcus Aureus ◽

Environmental Samples ◽

Pathogenic Bacteria ◽

Bacterial Species ◽

Raw Meat ◽

Food Borne Pathogens ◽

Food Borne ◽

Shigella Species ◽

Wide Range ◽

Meat Samples

Background: This study was conducted to examine the frequency of contamination in retail meat available in Karachi, Pakistan. Methodology: Raw meat samples (250) and surface swabs (90) from meat processing equipment and the surrounding environment were analyzed for microbiological contamination. Results: Out of 340 samples, 84% were found to be contaminated with bacterial species, including Klebsiella, Enterobacter, Staphylococcus aureus and Bacillus subtilis. A total of 550 (66%) of the bacterial isolates were potential pathogens. Of these, 342 and 208 isolates were from meat and environmental samples respectively. Food-borne pathogens isolated from meat samples included Escherichia coli O157:H7, Listeria, Salmonella Enteritidis and Shigella species whereas environmental samples yielded Staphylococcus aureus and Shigella species. Four strains of Brucella species were also isolated from meat samples. Total aerobic counts ranged between 108 -1010 CFU/g or cm2. Resistance to a wide range of antibiotics was observed. Resistance rates to ampicillin, amoxicillin, novobiocin and cefaclor were from 62 to 75% in general. Thirty-three percent of Salmonella isolates were resistant to ampicillin. No quinolone resistance was observed. Biofilm formation was observed among 88 (16%) pathogenic bacteria including E. coli, Klebsiella, Enterobacter species and Staphylococcus aureus. Conclusions: Food-borne pathogens found in retail shops could be sources for horizontal contamination of meat. Our data confirm the circulation of antibiotic resistant and biofilm forming pathogens in raw meat and its environment in retail shops in Pakistan, which could play a role in the spread of antimicrobial resistance amongst food-borne bacteria.

Download Full-text

A Rapid and Sensitive Europium Nanoparticle-Based Lateral Flow Immunoassay Combined with Recombinase Polymerase Amplification for Simultaneous Detection of Three Food-Borne Pathogens

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph18094574 ◽

2021 ◽

Vol 18 (9) ◽

pp. 4574

Author(s):

Kai Chen ◽

Biao Ma ◽

Jiali Li ◽

Erjing Chen ◽

Ying Xu ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Vibrio Parahaemolyticus ◽

Pathogenic Bacteria ◽

Simultaneous Detection ◽

Lateral Flow ◽

Recombinase Polymerase Amplification ◽

Quantitative Detection ◽

Bacterial Strains ◽

Food Borne Pathogens ◽

Food Borne

Food-borne pathogens have become an important public threat to human health. There are many kinds of pathogenic bacteria in food consumed daily. A rapid and sensitive testing method for multiple food-borne pathogens is essential. Europium nanoparticles (EuNPs) are used as fluorescent probes in lateral flow immunoassays (LFIAs) to improve sensitivity. Here, recombinase polymerase amplification (RPA) combined with fluorescent LFIA was established for the simultaneous and quantitative detection of Listeria monocytogenes, Vibrio parahaemolyticus, and Escherichia coliO157:H7. In this work, the entire experimental process could be completed in 20 min at 37 °C. The limits of detection (LODs) of EuNP-based LFIA–RPA were 9.0 colony-forming units (CFU)/mL for Listeria monocytogenes, 7.0 CFU/mL for Vibrio parahaemolyticus, and 4.0 CFU/mL for Escherichia coliO157:H7. No cross-reaction could be observed in 22 bacterial strains. The fluorescent LFIA–RPA assay exhibits high sensitivity and good specificity. Moreover, the average recovery of the three food-borne pathogens spiked in food samples was 90.9–114.2%. The experiments indicate the accuracy and reliability of the multiple fluorescent test strips. Our developed EuNP-based LFIA–RPA assay is a promising analytical tool for the rapid and simultaneous detection of multiple low concentrations of food-borne pathogens.

Download Full-text

Application of DNA microarray technology for detection, identification, and characterization of food-borne pathogens

Canadian Journal of Microbiology ◽

10.1139/w05-105 ◽

2006 ◽

Vol 52 (1) ◽

pp. 1-8 ◽

Cited By ~ 48

Author(s):

M Kostrzynska ◽

A Bachand

Keyword(s):

Dna Microarray ◽

Dna Sequences ◽

Pathogenic Bacteria ◽

Dna Microarrays ◽

Simultaneous Detection ◽

Microarray Technology ◽

Food Borne Pathogens ◽

Food Borne ◽

Target Dna

DNA microarrays represent the latest advance in molecular technology. In combination with bioinformatics, they provide unparalleled opportunities for simultaneous detection of thousands of genes or target DNA sequences and offer tremendous potential for studying food-borne microorganisms. This review provides an up-to-date look at the application of DNA microarray technology to detect food-borne pathogenic bacteria, viruses, and parasites. In addition, it covers the advantages of using microarray technology to further characterize microorganisms by providing information for specific identification of isolates, to understand the pathogenesis based on the presence of virulence genes, and to indicate how new pathogenic strains evolved epidemiologically and phylogenetically.Key words: DNA microarrays, food-borne pathogens, detection.

Download Full-text

Epitheliocystis: Development of PCR assay for the monitoring among the commercially important aquaculture species of Ukraine

Regulatory Mechanisms in Biosystems ◽

10.15421/021932 ◽

2019 ◽

Vol 10 (2) ◽

pp. 215-218

Author(s):

V. K. Zezekalo ◽

S. B. Peredera ◽

K. F. Pochernayev ◽

M. A. Petrenko ◽

P. P. Shatokhin ◽

...

Keyword(s):

Veterinary Medicine ◽

Bacterial Species ◽

Simultaneous Detection ◽

Ctenopharyngodon Idella ◽

Gibel Carp ◽

Epithelial Tissue ◽

Pcr Assay ◽

Thermo Fisher Scientific ◽

Pcr Products ◽

Commercially Important

Epitheliocystis is an emerging disease of wild and cultured fish caused by a number of bacterial species, characterized by the presence of cytoplasmic bacterial inclusions in the epithelial cells of the gills, which contribute to the merging of the gill plates, and in some cases also spread to the skin of fish. This disease may manifest as hypertrophy and inflammation of the gills, white nodular lesions of epithelial tissue in the gills or skin, gasping on the surface of the water, lethargy, poor swimming and stunted growth. Among the commercially important aquaculture species of Ukraine, such as Atlantic salmon (Salmo salar), brown trout (S. trutta), grass carp (Ctenopharyngodon idella), common carp (Cyprinus carpio) and gibel carp (Carassius auratus), Candidatus Clavochlamydia salmonicola and Candidatus Piscichlamydia salmonis are associated with epitheliocystis. There are currently no tools at the disposal of ichthyologists and veterinary laboratories in Ukraine to identify Ca. C. salmonicola and Ca. Piscichlamydia salmonis. Our basic concern was to develop a PCR assay of epitheliocystis diagnosis. We suggest the use of general primers for simultaneous detection of Ca. C. salmonicola and Ca. Piscichlamydia salmonis. The developed PCR assay for identification of Ca. C. salmonicola and Ca. Piscichlamydia salmonis has shown its suitability for amplifying control DNA. Confirmation of the amplification products identity was performed using selective recognition of the sequence by the TasI restriction endonuclease (Thermo Fisher Scientific, US). Analytical specificity verification of the PCR assay performed by amplifying the control DNA of 10 species of the Chlamydiales order showed the absence of PCR products, but observed in one. The designed PCR assay, after approbation on clinical material, can be used by researchers for extensive monitoring of epitheliocystis, doctors of veterinary medicine for diagnosis clarification, in addition to introduction into the practice of veterinary medicine laboratories and implementation in fish farm improvement programmes. The amplicon size of 197 base pairs theoretically permits application of this oligonucleotide primers pair for real-time PCR.

Download Full-text

Development of a DNA microarray assay for rapid detection of fifteen bacterial pathogens in pneumonia

10.21203/rs.2.24314/v3 ◽

2020 ◽

Author(s):

Xiuqing Ma ◽

Yanqin Li ◽

Yuan Liang ◽

Yang Liu ◽

Ling Yu ◽

...

Keyword(s):

Dna Microarray ◽

Legionella Pneumophila ◽

Burkholderia Cepacia ◽

Pathogenic Bacteria ◽

Chlamydia Pneumoniae ◽

Bacterial Pathogens ◽

Bacterial Species ◽

Simultaneous Detection ◽

Clinical Isolates ◽

Microarray Assay

Abstract Background: The rapid identification of pathogenic bacteria is important for determining an appropriate antimicrobial therapy for pneumonia, but traditional bacterial culture is time-consuming and labourious. The aim of this study was to develop and evaluate a DNA microarray assay for the simultaneous detection of fifteen bacterial species directly from respiratory tract specimens in patients with pneumonia. These species included Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Mycoplasma pneumoniae, Enterococcus faecalis, Enterococcus faecium, Enterobacter cloacae, Stenotrophomonas maltophilia, Burkholderia cepacia, Legionella pneumophila and Chlamydia pneumoniae. The 16S rDNA genes and other specific genes of each pathogen were chosen as the amplification targets, amplified via multiplex polymerase chain reaction (PCR), and hybridized to oligonucleotide probes in a microarray.Results: The DNA microarray detection limit was 103 copies/μL. Nineteen standard strains and 119 clinical isolates were correctly detected with our microarray, and 3 nontarget species from 4 clinical isolates were not detected. Additionally, bacterial pathogens were accurately identified when two or three bacterial targets were mixed together. Furthermore, the results for 99.4% (156/157) of clinical specimens were the same as those from a conventional assay.Conclusions: We developed a DNA microarray that could simultaneously detect various bacterial pathogens in pneumonia. The method described here has the potential to provide considerable labour and time savings due to its ability to screen for 15 bacterial pathogens simultaneously.

Download Full-text

Pathogenic Bacterial Species in Locally Prepared Fresh Fruit Juices Sold in Juice Houses of Eastern Ethiopia

Microbiology Insights ◽

10.1177/11786361211060736 ◽

2021 ◽

Vol 14 ◽

pp. 117863612110607

Author(s):

Dechasa Adare Mengistu ◽

Negga Baraki ◽

Tesfaye Gobena Tesema

Keyword(s):

Pathogenic Bacteria ◽

Fruit Juice ◽

Statistical Tests ◽

Statistical Significance ◽

Bacterial Species ◽

Fruit Juices ◽

Fresh Fruit ◽

Plate Count ◽

Eastern Ethiopia ◽

Food Borne

Fruit juices are important components of a healthy diet and a dietary source of nutrients, vitamins, and fiber and vital for human health. However, unless it is handled with safety and hygienic conditions, food can be a vehicle for the transmission of various agents of diseases resulting in food borne outbreaks. Thus, this study aimed to determine common pathogenic bacterial species in locally prepared fresh fruit juices sold in juice houses of Eastern Ethiopia. A cross-sectional study design was conducted from 1 January to 27 March 2020, in eastern Ethiopia. Seventy-eight juice samples were collected aseptically using a sterilized collecting jar from each juice house. Pour plate count method was used to determine Staphylococcus, Salmonella, and Shigella species. Finally, the data were analyzed using descriptive statistical tests such as Chi-square and Fisher’s exact tests. P-value of .05 was considered as a cut point for statistical significance. The study found Staphylococcus count ranged from 1.68 log CFU/mL with the mean value of 4.204 log CFU/mL. Overall, 58 (74.4%) of the fruit juice samples had Staphylococcus count, 19 (24.4%) had Salmonella and 12 (15.4%) had Shigella higher than the maximum permitted limit of Gulf standard, 2000 set for any type of fruit juice. In general, the study found more than two-thirds of fruit juice samples had at least 1 pathogenic bacteria species higher than the maximum permitted limit and potentially hazardous to consumer health. Thus, regular supervision and application of food hygiene and safety are essential to improve the quality of fruit juice and to prevent the consumption of contaminated fruit juices, which leads to food borne illness.

Download Full-text

Synthesis and Antibacterial Activity of Rhodanine-Based Azo Dyes and Their Use as Spectrophotometric Chemosensor for Fe3+ Ions

Chemosensors ◽

10.3390/chemosensors8010016 ◽

2020 ◽

Vol 8 (1) ◽

pp. 16 ◽

Cited By ~ 4

Author(s):

Dana Akram ◽

Ismail A. Elhaty ◽

Shaikha S. AlNeyadi

Keyword(s):

Pathogenic Bacteria ◽

Biological Activities ◽

Bacterial Species ◽

High Sensitivity ◽

Azo Compounds ◽

Molar Ratio ◽

Spectroscopic Techniques ◽

Reference Drug ◽

Physiochemical Properties ◽

Design And Synthesis

This research includes the design and synthesis of new derivatives for rhodanine azo compounds (4a–c) containing a naphthalene ring. Physiochemical properties of the synthesized compounds were determined by their melting points, FTIR, 1H-NMR, 13C-NMR, and elemental analysis spectroscopic techniques. The biological activities of the newly prepared azo rhodanine compounds were evaluated against some pathogenic bacteria using three different bacterial species including (Escherichia coli., Pseudomonas aeruginosa, Staphylococcus aureus) and compared with amoxicillin as a reference drug. The results showed that our compounds have moderate-to-good vital activity against the mentioned pathogenic bacteria. The selectivity and sensitivity of the newly prepared rhodanine azo compounds with transition metals Co2+, Cu2+, Zn2+, Ni2+, and Fe3+ were studied using UV–vis and fluorescence spectroscopy techniques. Among the synthesized azos, azo 4c showed affinity toward Fe3+ ions with an association constant of 4.63 × 108 M−1. Furthermore, this azo showed high sensitivity toward Fe3+ ions with detection limits of 5.14 µM. The molar ratio and Benesi–Hildebrand methods confirmed the formation of complexes between azo 4c and Fe3+ with 1:2 binding stoichiometry. Therefore, azo 4c showed excellent potential for developing efficient Fe3+ chemosensors.

Download Full-text

Antibacterial Activity and Optimisation of Bacteriocin Producing Lactic Acid Bacteria Isolated from Beef (Red Meat) Samples

Biological Sciences - PJSIR ◽

10.52763/pjsir.biol.sci.59.2.2016.85.98 ◽

2016 ◽

Vol 59 (2) ◽

pp. 85-98

Author(s):

Saiqa Andleeb ◽

Nazish Mazhar Ali ◽

Bushra Mazhar ◽

Iram Khadija ◽

Bushra Kalim

Keyword(s):

Antibacterial Activity ◽

Lactic Acid ◽

Lactic Acid Bacteria ◽

Bacterial Species ◽

Meat Products ◽

Diffusion Method ◽

Bacterial Strains ◽

Biochemical Tests ◽

Food Borne ◽

Meat Samples

Bacteriocin producing bacteria are commonly found in meat products to enhance theirshelf-life. In the present study, bacterial species were isolated from meat samples (beef) from differentlocalities of Lahore, Pakistan. MRS agar medium was used to isolate lactic acid bacteria (LAB) throughspread and streak methods (incubated for 72 h at 37 °C). Identification of bacteriocinogenic LAB strainswas done by using staining techniques, morphology based characteristics and biochemical tests. Thesestrains were BSH 1b, BSH 3a, BIP 4a, BIP 3a, BIP 1b and BRR 3a. Antibacterial activity of LAB wasperformed against food borne pathogens viz., Escherichia coli and Staphylococcus aureus through paperdisc diffusion method. Three bacterial strains showed maximum inhibition and characterised by ribotypingviz., BIP 4a was identified as Lactobacillus curvatus, BIP 3a was Staphylococcus warneri and BIP 1b wasLactobacillus graminis. Optimum pH 5-6.5 and 30-37 °C temperature for isolated bacterial strains wasrecorded. Protein concentration measured was 0.07 mg/mL for BSH 1b, 0.065 mg/mL for BSH 3a,0.057 mg/mL for BIP 4a, 0.062 mg/mL for BIP 1b, 0.065 mg/mL for BIP 3a and for BRR 3a 0.078 mg/mL,respectively. Bacteriocin of all isolates except BIP 3a was found to be sensitive towards pepsin and resistanttowards Rnase. Bacteriocin production was stable at between pH 5.0 and 6.0 and resistant temperaturewas 40 °C. It was concluded that lactic acid bacteria (LAB) from meat can be helpful as antibacterialagents against food-borne bacterial pathogens because of thermostable producing bacteriocin.

Download Full-text

Bacterial causes of upper & lower respiratory tract infection in Sheep

The Iraqi Journal of Veterinary Medicine ◽

10.30539/iraqijvm.v29i1.858 ◽

2005 ◽

Vol 29 (1) ◽

pp. 1-10

Author(s):

Jamal Salman Ali

Keyword(s):

Tract Infection ◽

Respiratory Tract ◽

Nasal Cavity ◽

Respiratory Tract Infection ◽

Lower Respiratory Tract Infection ◽

Lower Respiratory Tract ◽

Pathogenic Bacteria ◽

Bacterial Species ◽

High Sensitivity ◽

Hemorrhagic Lesion

This study was designed to investigate the bacterial species that induceupper and lower respiratory tract infection in sheep, and to find out anyrelationship which may exist between them.Therefore two groups of sheep were employed. The first group wassuffering from certain respiratory signs. While the second group was apparentlyhealthy. Each group included 50 sheep. Research samples were collected for aperiod of six months from AL-Shulla Abattoir.Microbiological investigation indicated the isolation of certainmicroorganisms from all animals in both groups from nasal cavity, and from 34lungs of the first group and 16 lungs of the second group. The number of isolatesfrom the nasal cavity, were 200 from different species ,43 isolates from thebronchioles and 70 from the lungs tissue. On the other hand the number ofbacterial isolates from the nasal cavity, bronchioles and the lung tissue of thefirst group were 113, 29 and 55 respectively.The study revealed the isolation of potentially pathogenic bacteria from thelower respiratory system of both groups, these bacteria were namely Pasteurellahaemolytica of serotype (A2), Niesseria spp. and Corynebacterium pyogenes,the number of isolates were 6 for each, and 8 isolates of Staphylococcus aureus.These bacteria were also isolated from the nasal cavity. The isolation of thesebacteria from the nasal strongly suggested their presence in the lungs and theprobable role in lesion formation.Animal inoculation were performed to study the virulence of P.haemolytica which caused certain hemorrhagic lesion in the lung , liver andkidney, with areas of necrosis in the lungs of the experimentally inoculatedrabbit, and caused death in mice. While the inoculation of C. ovis caused thedeath of rabbits within 72 hours, together with the presence of multipleabscessation on the internal organs and abdomenal wall.Sensitivity tests indicated a high sensitivity of most isolates to Gentamicin,Erythromycin and Kanamycin.

Download Full-text

COV-ID: A LAMP sequencing approach for high-throughput co-detection of SARS-CoV-2 and influenza virus in human saliva

10.1101/2021.04.23.21255523 ◽

2021 ◽

Author(s):

Robert Warneford-Thomson ◽

Parisha P. Shah ◽

Patrick Lundgren ◽

Jonathan Lerner ◽

Benjamin S. Abella ◽

...

Keyword(s):

Influenza A ◽

Low Cost ◽

Diagnostic Testing ◽

Simultaneous Detection ◽

High Sensitivity ◽

Human Saliva ◽

Laboratory Equipment ◽

Sample Handling ◽

Co Detection ◽

Multiple Pathogens

ABSTRACTThe COVID-19 pandemic has created an urgent need for rapid, effective, and low-cost SARS-CoV-2 diagnostic testing. Here, we describe COV-ID, an approach that combines RT-LAMP with deep sequencing to detect SARS-CoV-2 in unprocessed human saliva with high sensitivity (5–10 virions). Based on a multi-dimensional barcoding strategy, COV-ID can be used to test thousands of samples overnight in a single sequencing run with limited labor and laboratory equipment. The sequencing-based readout allows COV-ID to detect multiple amplicons simultaneously, including key controls such as host transcripts and artificial spike-ins, as well as multiple pathogens. Here we demonstrate this flexibility by simultaneous detection of 4 amplicons in contrived saliva samples: SARS-CoV-2, influenza A, human STATHERIN, and an artificial SARS spike-in. The approach was validated on clinical saliva samples, where it showed 100% agreement with RT-qPCR. COV-ID can also be performed directly on saliva adsorbed on filter paper, simplifying collection logistics and sample handling.

Download Full-text