Multiplex Detection of Salmonella spp., E. coli O157 and L. monocytogenes by qPCR Melt Curve Analysis in Spiked Infant Formula

Food poisoning continue to be a threat in the food industry showing a need to improve the detection of the pathogen responsible for the hospitalization cases and death. DNA-based techniques represent a real advantage and allow the detection of several targets at the same time, reducing cost and time of analysis. The development of new methodology using SYBR Green qPCR for the detection of L. monocytogenes, Salmonella spp. and E. coli O157 simultaneously was developed and a non-competitive internal amplification control (NC-IAC) was implemented to detect reaction inhibition. The formulation and supplementation of the enrichment medium was also optimized to allow the growth of all pathogens. The limit of detection (LoD) 95% obtained was <1 CFU/25 g for E. coli O157, and 2 CFU/25 g for Salmonella spp. and L. monocytogenes and regarding the multiplex detection a LoD 95% of 1.7 CFU/25 g was observed. The specificity, relative sensitivity and accuracy of full methodology were 100% and the use of the NC-IAC allowed the reliability of the results without interfering with the sensitivity of the methodology. The described study proved to obtain results comparable to those of probe-based qPCR, and more economically than classical high resolution melting qPCR, being both important aspects for its implementation in the food industry.

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Correction: Azinheiro et al. Multiplex Detection of Salmonella spp., E. coli O157 and L. monocytogenes by qPCR Melt Curve Analysis in Spiked Infant Formula. Microorganisms 2020, 8, 1359

Microorganisms ◽

10.3390/microorganisms9071482 ◽

2021 ◽

Vol 9 (7) ◽

pp. 1482

Author(s):

Sarah Azinheiro ◽

Joana Carvalho ◽

Marta Prado ◽

Alejandro Garrido-Maestu

Keyword(s):

Infant Formula ◽

Curve Analysis ◽

Multiplex Detection ◽

Salmonella Spp ◽

Melt Curve Analysis ◽

E Coli ◽

Published Paper

The authors would like to make the following correction to the published paper [...]

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Development of an Immunoassay for Detection of Staphylococcal Enterotoxin-Like J, A Non-Characterized Toxin

Toxins ◽

10.3390/toxins10110458 ◽

2018 ◽

Vol 10 (11) ◽

pp. 458 ◽

Cited By ~ 1

Author(s):

Hisaya Ono ◽

Nobuaki Hachiya ◽

Yasunori Suzuki ◽

Ikunori Naito ◽

Shouhei Hirose ◽

...

Keyword(s):

Limit Of Detection ◽

Expression System ◽

Sandwich Elisa ◽

Food Poisoning ◽

High Sensitivity ◽

Enzyme Linked Immunosorbent Assay ◽

E Coli ◽

Detection Systems ◽

C Terminus ◽

Culture Supernatants

Staphylococcal enterotoxins (SEs) are the cause of staphylococcal food poisoning (SFP) outbreaks. Recently, many new types of SEs and SE-like toxins have been reported, but it has not been proved whether these new toxins cause food poisoning. To develop an immunoassay for detection of SE-like J (SElJ), a non-characterized toxin in SFP, a mutant SElJ with C-terminus deletion (SElJ∆C) was expressed and purified in an E. coli expression system. Anti-SElJ antibody was produced in rabbits immunized with the SElJ∆C. Western blotting and sandwich enzyme-linked immunosorbent assay (ELISA) detection systems were established and showed that the antibody specifically recognizes SElJ without cross reaction to other SEs tested. The limit of detection for the sandwich ELISA was 0.078 ng/mL, showing high sensitivity. SElJ production in S. aureus was detected by using the sandwich ELISA and showed that selj-horboring isolates produced a large amount of SElJ in the culture supernatants, especially in that of the strain isolated from a food poisoning outbreak in Japan. These results demonstrate that the immunoassay for detection of SElJ is specific and sensitive and is useful for determining the native SElJ production in S. aureus isolated from food poisoning cases.

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Low Prevalence of Salmonella and Shiga Toxin–Producing Escherichia coli in Lymph Nodes of Australian Beef Cattle

Journal of Food Protection ◽

10.4315/0362-028x.jfp-17-180 ◽

2017 ◽

Vol 80 (12) ◽

pp. 2105-2111 ◽

Cited By ~ 3

Author(s):

Gavin Bailey ◽

Long Huynh ◽

Lachlan Govenlock ◽

David Jordan ◽

Ian Jenson

Keyword(s):

Escherichia Coli ◽

Lymph Nodes ◽

Shiga Toxin ◽

Limit Of Detection ◽

Ground Beef ◽

Plate Count ◽

Salmonella Spp ◽

Live Animal ◽

E Coli ◽

Low Prevalence

ABSTRACT Salmonella contamination of ground beef has been viewed as originating from the surface of carcasses. Recent studies have identified lymph nodes as a potential source of Salmonella contamination because these tissues play an active role in containment of pathogens in the live animal and because some lymph nodes are unavoidably present in manufacturing beef trimmings or primal cuts that may be incorporated into ground beef. A survey was conducted of the microbiological status of lymph nodes from Australian cattle at the time of slaughter to determine the prevalence of microbiological contamination. Sets of lymph nodes (n = 197), consisting of the superficial cervical (prescapular), prepectoral, axillary, presternal, popliteal, ischiatic, subiliac (precrural), coxalis, and iliofemoralis (deep inguinal), were collected from five geographically separated Australian abattoirs over a period of 14 months. Samples were tested for the presence of Salmonella spp. and Shiga toxin–producing Escherichia coli by BAX PCR assay. Aerobic plate count, E. coli, and coliforms were enumerated with a lower limit of detection of 80 CFU per node. The observed prevalence of Salmonella within peripheral lymph nodes was 0.48% (7 of 1,464). Two of the seven lymph nodes in which Salmonella organisms were detected came from the same animal. Grass-fed, grain-fed, and cull dairy cattle were all found to have detectable Salmonella in lymph nodes. All Salmonella detections occurred during cooler months of the year. No Shiga toxin–producing E. coli were detected. Aerobic microorganisms were detected above the limit of quantification in 3.2% of nodes (median count 2.24 log per node), and E. coli was detected in 0.8% of nodes (median count 3.05 log per node). The low prevalence of Salmonella and low concentration of aerobic microorganisms in Salmonella-positive lymph nodes of Australian cattle at the time of slaughter suggest that the likelihood of lymph nodes contributing significantly to the presence of Salmonella in ground beef is low.

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Persistence of Human Pathogens in Manure-Amended Australian Soils Used for Production of Leafy Vegetables

Agriculture ◽

10.3390/agriculture11010014 ◽

2020 ◽

Vol 11 (1) ◽

pp. 14

Author(s):

Jennifer Ekman ◽

Adam Goldwater ◽

Mark Bradbury ◽

Jim Matthews ◽

Gordon Rogers

Keyword(s):

Production Systems ◽

Limit Of Detection ◽

Probability Of Detection ◽

Enteric Pathogens ◽

Leafy Vegetables ◽

Vegetable Production ◽

Human Pathogens ◽

Salmonella Spp ◽

E Coli ◽

Vegetable Farm

Incorporation of manures into soil can add nutrients, improve soil structure and enhance biodiversity, thereby improving the sustainability of vegetable production systems. Unfortunately, manures can also potentially contain human enteric pathogens. To reduce the risk of contamination, Australian guidelines recommend a withholding period of 90 days between manure application and harvest for high-risk products such as leafy salad greens. Our study examined the appropriateness of these guidelines under conditions replicating those on a commercial vegetable farm. Cow manure and poultry litter with/without addition of non-pathogenic strains of Escherichia coli (E. coli), Listeriainnocua and Salmonella sv. sofia were added to sandy and clay loam soils typical of those used to grow vegetables. Bacterial populations were monitored in the soil and on crops of cos lettuce during spring (A), summer (B) and autumn (C) trials, with testing conducted by a commercial laboratory. Significant declines in E. coli occurred within 6 to 16 days in all trials. Modelling indicated that E. coli populations would be at or close to the limit of detection within 50 days for all of the combinations tested. A 2–3 log die-off of Salmonella spp. occurred within three weeks. However, occasional detections continued throughout trial A. As a result, the probability of detection after 50 days fell from 1.0 to 0.1 and 0.02 in trials B and C, respectively, but remained at 0.44 in trial A. Listeria spp. was the most persistent in soil but was not detected on lettuce at commercial maturity. While this study was limited in scope, the results suggest that a 90 day withholding period between application of manure and harvest significantly reduces risk from enteric pathogens under Australian field conditions.

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A newly developed polydopamine-coated paper-based microchip for rapid and highly selectivity detection of foodborne pathogens

10.21203/rs.3.rs-150586/v1 ◽

2021 ◽

Author(s):

Jinfeng Liu ◽

Mimi Zhang ◽

Zhiqiang Shen ◽

Yongxin Liu ◽

Yang Song ◽

...

Keyword(s):

Foodborne Pathogens ◽

Pathogen Detection ◽

Lower Cost ◽

Limit Of Detection ◽

Fluorescence Labeling ◽

Economic Losses ◽

Great Promise ◽

Salmonella Spp ◽

Coated Paper ◽

E Coli

Abstract Background In 2020, Covid-19 pneumonia has had a great impact on human health in although the countries around the world, it brings serious threaten to people’s lives and resulted in serious economic losses. At the same time, a lot news about the detection of Covid-19 in food emerges endlessly, a rapid and high selectivity detection method or technology is in urgent need for its ability to help relevant departments effectively control the epidemic situation and ensuring people’s lives and property safety. In recent years, loop-mediated isothermal amplification (LAMP) has been certified as a quick and highly selective technique to detect foodborne microorganisms. Results In this paper, a newly developed microchip with polydopamine-coated paper based on LAMP was fabricated. This microchip consists of nine chambers for sampling and reactions, the targeted nucleic acid of foodborne pathogens was labeled by calcein fluorescence rather than SYBR. The microchip is advantageous of lower cost of materials and simple pretreated methods, and is easy to operate without the need for complex controlled fluid flow. The LAMP procedure and fluorescence detection of pathogens can be carried on the chip without opening the lid, preventing aerosol contamination and reducing the probability of false positives. In experiments, the LAMP reaction conditions including the optimal reaction temperature and reaction time are thoroughly discussed and have been executed for various foodborne bacteria samples, including Escherichia coli O157:H7 (E. coli O157:H7), Salmonella spp., Staphylococcus aureus (S. aureus), and Vibrio parahaemolyticus (V. parahaemolyticus). Testing of E. coli O157:H7 proved to be highly selective and sensitive (as low as 0.0134 ng µL− 1). Additionally, experimental test of real milk sample was figured, the complete detection duration time was within 68 min, the limit of detection(LOD) for Salmonella spp. was determined to be lower than 12 CFU mL− 1. Conclusion In summary, a newly developed LAMP microchip with polydopamine-coated and calcein fluorescence labeling paper-based provides a lower cost, easy to use, highly selective, and multiplexable pathogen detection capability with great promise as a rapid, highly efficient, and economical solution for future foodborne pathogen testing.

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Microbiological evaluation of constructed wetlands and solar disinfection in wastewater treatment and reuse

Journal of Water and Health ◽

10.2166/wh.2020.301 ◽

2020 ◽

Vol 18 (6) ◽

pp. 1146-1153

Author(s):

Vinícius B. Santos ◽

Beatriz S. Machado ◽

Alexandre Atalla ◽

Priscila S. Cavalheri ◽

Fernando J. C. Magalhães Filho

Keyword(s):

Constructed Wetlands ◽

Limit Of Detection ◽

Septic Tank ◽

Salmonella Spp ◽

Fecal Indicator ◽

Total Coliforms ◽

Solar Disinfection ◽

E Coli ◽

Significant Difference ◽

Shigella Spp

Abstract The objective of this study was to evaluate the removal of fecal indicator bacteria and select bacterial pathogens (total coliforms, Escherichia coli, Shigella spp., Salmonella spp. and Pseudomonas aeruginosa) in vertical flow constructed wetlands (VF-CWs) with earthworms and solar disinfection (SODIS) as post-treatment of effluent from a septic tank. There was no significant difference between them in removing bacteria using a VF-CW with earthworms (VF-CW W/E) and without earthworms (VF-CW N/E). Both VF-CWs did not completely remove pathogens; however, with the SODIS technology receiving the effluent from VF-CW W/E, for E. coli, Shigella spp., Salmonella spp. and P. aeruginosa, the values were reduced below the limit of detection and 4.3 log unit average reduction for total coliforms, with 6 h in SODIS.

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Microbiological Survey of Wheat Flour Sold at Retail in Canada, 2018-2019

Journal of Food Protection ◽

10.4315/jfp-20-297 ◽

2020 ◽

Author(s):

helen zhang ◽

Etsuko Yamamoto ◽

Johanna Murphy ◽

Catherine Carrillo ◽

Kate Hardie ◽

...

Keyword(s):

Escherichia Coli ◽

Wheat Flour ◽

Virulence Genes ◽

Limit Of Detection ◽

Mitigation Strategies ◽

Indicator Organisms ◽

Salmonella Spp ◽

Total Coliforms ◽

E Coli ◽

Two Samples

Following two O121 STEC outbreaks linked to wheat flour, this study was conducted to gain baseline information on the occurrence of bacterial pathogens and levels of indicator organisms in wheat flour in Canada. A total of 347 pre-packaged wheat flour samples were analyzed for Salmonella spp., Shiga Toxin-Producing Escherichia coli (STEC), Listeria monocytogenes ( L. monocytogenes ) , aerobic colony count (ACC), total coliforms, and generic Escherichia coli ( E. coli ) . Salmonella spp. and O157 STEC were not detected in any of the samples. L. monocytogenes was identified in two samples (0.6%) at levels below the limit of detection (<0.7 log CFU/g). Non-O157 STEC were isolated from six samples (1.7%) and were characterized for the presence of STEC virulence genes: stx 1, stx 2 and subtypes, eae , hlyA, and aggR . One O103:H25 STEC isolate carried virulence genes ( stx 1 a + eae ) that are known to be capable of causing diarrhea and/or bloody diarrhea in humans. Of the five remaining non-O157 STEC isolates, four carried single stx 2a or stx 2c genes and were considered to have the potential of causing diarrhea. The remaining non-O157 STEC isolate ( stx 2 ), while not a priority non-O157 STEC was not available for sequencing and thus its potential to cause illness is unknown. ACC, total coliforms, and generic E. coli were detected in 98.8%, 72.6% and 0.6% of the flour samples. The mean counts of ACC were greater in whole-wheat flour as compared to the other flour types tested ( p <0.001). The results of this study suggest that the occurrence of O157 STEC and Salmonella is low, but the occurrence of non-O157 STEC in wheat flour with the potential to cause human illness of diarrhea is relatively common. Therefore, the consumption of raw flour could increase the likelihood of STEC infections. Further research is merited for potential risk mitigation strategies within the food production system and with consumers.

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Surveillance of Antibiotic Resistance Among Enterobacteriaceae Strains Isolated in an Infectious Diseases Hospital from Romania

Revista de Chimie ◽

10.37358/rc.18.5.6298 ◽

2018 ◽

Vol 69 (5) ◽

pp. 1240-1243

Author(s):

Manuela Arbune ◽

Mioara Decusara ◽

Luana Andreea Macovei ◽

Aurelia Romila ◽

Alina Viorica Iancu ◽

...

Keyword(s):

Antibiotic Resistance ◽

Infectious Diseases ◽

Salmonella Spp ◽

Resistance Level ◽

E Coli ◽

Bacterial Genus ◽

Antibiotic Resistance Profile ◽

Carbapenem Resistant ◽

Invasive Infections ◽

Klebsiella Spp

The aim of the present study was to characterize the antibiotic resistance profile of enterobacteriaceae strains isolated in Infectious Diseases Hospital Galati, Romania, during 2016, in order to guide the local antibiotic stewardship strategy. There are 597 biological samples with positive cultures for enterobacteriaceae, related to invasive and non-invasive infections. The main bacterial genus were E. coli 62%, Klebsiella spp 15%, Proteus spp 11% and Salmonella spp 6%. Over a half of isolated strains have one or more antibiotic resistance. The resistance level depends on bacterial genus, with highest level found among the rare isolates: Enterobacter spp, Citrobacter spp, Morganella spp and Serratia spp. The rate of MDR was 17.,6% for E. coli, 40.9% for Klebsiella spp and 50.7% for Proteus spp. while the rate of strains producing Extended Spectrum of Beta Lactamase are 7.2% for E. coli, 28.4% for Klebsiella spp and 12.3% for Proteus spp. The carbapenem resistant strains were found in 1.1% cases.

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Antimicrobial Efficacy and Spectrum of Phosphorous-Fluorine Co-Doped TiO2 Nanoparticles on the Foodborne Pathogenic Bacteria Campylobacter jejuni, Salmonella Typhimurium, Enterohaemorrhagic E. coli, Yersinia enterocolitica, Shewanella putrefaciens, Listeria monocytogenes and Staphylococcus aureus

Foods ◽

10.3390/foods10081786 ◽

2021 ◽

Vol 10 (8) ◽

pp. 1786

Author(s):

György Schneider ◽

Bettina Schweitzer ◽

Anita Steinbach ◽

Botond Zsombor Pertics ◽

Alysia Cox ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Campylobacter Jejuni ◽

Food Industry ◽

Pathogenic Bacteria ◽

Antibacterial Activities ◽

Tio2 Nps ◽

E Coli ◽

Foodborne Pathogenic Bacteria ◽

And Staphylococcus Aureus ◽

Co Doped

Contamination of meats and meat products with foodborne pathogenic bacteria raises serious safety issues in the food industry. The antibacterial activities of phosphorous-fluorine co-doped TiO2 nanoparticles (PF-TiO2) were investigated against seven foodborne pathogenic bacteria: Campylobacter jejuni, Salmonella Typhimurium, Enterohaemorrhagic E. coli, Yersinia enterocolitica, Shewanella putrefaciens, Listeria monocytogenes and Staphylococcus aureus. PF-TiO2 NPs were synthesized hydrothermally at 250 °C for 1, 3, 6 or 12 h, and then tested at three different concentrations (500 μg/mL, 100 μg/mL, 20 μg/mL) for the inactivation of foodborne bacteria under UVA irradiation, daylight exposure or dark conditions. The antibacterial efficacies were compared after 30 min of exposure to light. Distinct differences in the antibacterial activities of the PF-TiO2 NPs, and the susceptibilities of tested foodborne pathogenic bacterium species were found. PF-TiO2/3 h and PF-TiO2/6 h showed the highest antibacterial activity by decreasing the living bacterial cell number from ~106 by ~5 log (L. monocytogenes), ~4 log (EHEC), ~3 log (Y. enterolcolitca, S. putrefaciens) and ~2.5 log (S. aureus), along with complete eradication of C. jejuni and S. Typhimurium. Efficacy of PF-TiO2/1 h and PF-TiO2/12 h NPs was lower, typically causing a ~2–4 log decrease in colony forming units depending on the tested bacterium while the effect of PF-TiO2/0 h was comparable to P25 TiO2, a commercial TiO2 with high photocatalytic activity. Our results show that PF-co-doping of TiO2 NPs enhanced the antibacterial action against foodborne pathogenic bacteria and are potential candidates for use in the food industry as active surface components, potentially contributing to the production of meats that are safe for consumption.

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Incidence and characterisation of Bacillus cereus bacteriophages from Thua Nao, a Thai fermented soybean product

BioMolecular Concepts ◽

10.1515/bmc-2021-0009 ◽

2021 ◽

Vol 12 (1) ◽

pp. 85-93

Author(s):

Wallapat Phongtang ◽

Ekachai Chukeatirote

Keyword(s):

Bacillus Cereus ◽

Food Industry ◽

Pathogenic Bacteria ◽

Morphological Analysis ◽

Food Poisoning ◽

Fermented Soybean ◽

Foodborne Pathogenic Bacteria ◽

Ph Range ◽

Potential Use

Abstract Bacillus cereus is considered to be an important food poisoning agent causing diarrhea and vomiting. In this study, the occurrence of B. cereus bacteriophages in Thai fermented soybean products (Thua Nao) was studied using five B. cereus sensu lato indicator strains (four B. cereus strains and one B. thuringiensis strain). In a total of 26 Thua Nao samples, there were only two bacteriophages namely BaceFT01 and BaceCM02 exhibiting lytic activity against B. cereus. Morphological analysis revealed that these two bacteriophages belonged to the Myoviridae. Both phages were specific to B. cereus and not able to lyse other tested bacteria including B. licheniformis and B. subtilis. The two phages were able to survive in a pH range between 5 and 12. However, both phages were inactive either by treatment of 50°C for 2 h or exposure of UV for 2 h. It should be noted that both phages were chloroform-insensitive, however. This is the first report describing the presence of bacteriophages in Thua Nao products. The characterization of these two phages is expected to be useful in the food industry for an alternative strategy including the potential use of the phages as a biocontrol candidate against foodborne pathogenic bacteria.

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