isolation and purification of campylobacter jejuni and campylobacter coli from raw chicken

Isolamento potenziato del Campylobacter dalla carne

Campylobacter spp. are the leading cause of bacterial foodborne gastroenteritis worldwide, with an estimated 800 million cases annually1. As a major zoonotic bacterium, Campylobacter naturally colonizes the gastrointestinal tracts of a wide range of animals, including wild birds, farm animals, and pets2. During slaughtering or food processing, Campylobacter spp. frequently contaminate carcasses or meat products3. Campylobacteriosis is usually associated with the consumption of undercooked poultry or cross-contamination of other foods by raw poultry juices2. It can cause serious complications, such as Guillain-Barr&#233; syndrome, reactive arthritis, and septicemia in immunocompromised individuals4. Detecting and isolating Campylobacter from food sources, especially poultry products, is essential for public health surveillance, outbreak investigation, and risk assessment.
Conventional culture-based methods are the traditional and standard methods for Campylobacter detection5,6. However, there are several limitations, including long incubation times (48 h or more), low sensitivity (up to 50%), and are not inclusive to all strains (some stressed Campylobacter cells may not grow well or at all in the media)7. Molecular methods, such as polymerase chain reaction (PCR), are more rapid and sensitive than culture-based methods, but they do not provide viable isolates for further characterization8,9.
Immunological methods are alternative and complementary methods for Campylobacter detection. These are rapid, simple, and versatile, but also have several limitations, including cross-reactivity (some antibodies may bind to non-Campylobacter bacteria or other substances that share similar antigens), low specificity (some antibodies may not bind to all Campylobacter strains or serotypes), and sample preparation requirements (immunological methods often require pre-treatment of the samples to remove interfering substances to enhance the binding of the antibodies)10.
Within the genus of Campylobacter, C. jejuni and C. coli cause most human Campylobacter infections (81% and 8.4%, respectively)11. Both are spiral-shaped, microaerophilic, and thermophilic bacteria containing a unipolar flagellum or bipolar flagella. Rotation of a flagellum at each pole is considered both the primary driving force for its characteristic corkscrew motility and crucial to its pathogenesis because it allows the bacterium to swim through the viscous mucosa of the host gastrointestinal tract. The motility of Campylobacter is controlled by its chemosensory system that allows the cells to move toward favorable environments12,13. Based on the cell morphology and physiological characteristics of Campylobacter, a few studies have utilized membrane filtration for the isolation of Campylobacter spp. from fecal and environmental samples14,15,16.
This study presents a rapid and robust protocol for the isolation and subsequent detection of C. jejuni and C. coli from raw meat, which overcomes the drawbacks of the existing methods and offers several advantages. Tentative colonies can be confirmed as Campylobacter spp. using a variety of methods, such as microscopy, biochemical tests (e.g., catalase and oxidase activity assays), or molecular methods6. The method identifies the isolates at the species level using a multiplex real-time PCR (mqPCR) assay that targets genes unique to C. jejuni and C. coli. This method is relatively inexpensive, rapid, and selective, which makes it suitable for use in a variety of settings, including food processing facilities, clinical laboratories, and research laboratories.

Autore in primo piano: Sviluppo di un protocollo avanzato per l'isolamento rapido e accurato del Campylobacter dai prodotti alimentari

Rilevamento e isolamento di Campylobacter spp. da carne cruda

This study provides a rapid and robust protocol for the isolation of Campylobacter from food products. It overcomes several drawbacks of existing methods and offers advantages for the subsequent detection and characterization of isolate. Rinse aids in the field are often 10, 000 times larger than the samples used in food safety detection assays, such as PCR.There's a need to reduce the error of sub-sampling in order to ensure the results are representative of the population of interest. This protocol has been used to isolate over 50 strains of Campylobacter, which have subsequently been utilized in genomic studies. Our data and analysis contributed to understanding the molecule mechanisms of Campylobacter host agents and horizontal gene transfer.Such in size will facilitate the development of novel therapeutic strategies for managing Campylobacterosis. This protocol offers the opportunity to reduce sub sampling by a factor of 13 and also reduces the time required to isolate Campylobacter from raw meat by 24 hours or within an eight hour workday. Future studies can apply this methodology for the isolation of Campylobacter, not only from food products, but from environmental samples as well.This can help eliminate false negative results and provide more accurate quantification of the number of contaminated samples.

Isolamento e purificazione di Campylobacter jejuni e Campylobacter coli da pollo crudo

Per iniziare, posizionare le piastre di agar Brucella con i coperchi aperti in un armadietto di sicurezza biologica per l'essiccazione. Posizionare un filtro in acetato di cellulosa al centro di una piastra di agar Brucella. Pipettare quattro gocce per filtro e 20 microlitri per goccia di campione arricchito con Campylobacter sul filtro.Dopo aver incubato le gocce a temperatura ambiente per 15 minuti, rimuovere con cura i filtri, quindi incubare le piastre a 42 gradi Celsius per circa 24 ore. Dopo l'incubazione, prelevare le colonie di Campylobacter caratteristiche con tratti specifici. Striare le colonie su piastre di agar Brucella per la purificazione.Per la conservazione a lungo termine, raccogli le colonie di Campylobacter cresciute su piastre di agar Brucella e inocula il brodo Bolton. Posizionare i campioni all'interno di un contenitore sigillato che mantenga una miscela di gas composta dall'85% di azoto, dal 10% di anidride carbonica e dal 5% di ossigeno. Incubare i campioni a 42 gradi Celsius per una notte.Quindi aggiungere 900 microlitri della coltura di Campylobacter coltivata durante la notte a un crioviale da due millilitri contenente 100 microlitri di DMSO. Raffreddare rapidamente in un bagno di etanolo con ghiaccio secco per 10 minuti e trasferire in un congelatore a meno 80 gradi Celsius. Il confronto tra membrane filtranti di diverse dimensioni dei pori ha rivelato che più cellule sono state in grado di passare attraverso il filtro da 0,65 micrometri rispetto al filtro da 0,45 micrometri.

isolation-purification-campylobacter-jejuni-campylobacter-coli-from

Research

JoVE Journal

Biology

Isolation and Purification of Campylobacter jejuni and Campylobacter coli from Raw Chicken

191 Views.  Agricultural Research Service (ARS), United States Department of Agriculture (USDA). Per iniziare, posizionare le piastre di agar Brucella con i coperchi aperti in un armadietto di sicurezza biologica per l'essiccazione. Posizionare un filtro in acetato di cellulosa al centro di una piastra di agar Brucella. Pipettare quattro gocce per filtro e 20 microlitri per goccia di campione arricchito con Campylobacter sul filtro.Dopo aver incubato le gocce a temperatura ambiente per 15 minuti, rimuovere con cura i filtri, quindi incubare le piastre a 42 gradi Celsius per circa ...

Video: Isolamento e purificazione di Campylobacter jejuni  e Campylobacter coli  da pollo crudo

Detection and Isolation of Campylobacter spp. from Raw Meat

This article presents a rapid yet robust protocol for isolating Campylobacter spp. from raw meats, specifically focusing on Campylobacter jejuni and Campylobacter coli. The protocol builds upon established methods, ensuring compatibility with the prevailing techniques employed by regulatory bodies such as the Food and Drug Administration (FDA) and the U.S. Department of Agriculture (USDA) in the USA, as well as the International Organization for Standardization (ISO) in Europe. Central to this protocol is collecting a rinsate, which is concentrated and resuspended in Bolton Broth media containing horse blood. This medium has been proven to facilitate the recovery of stressed Campylobacter cells and reduce the required enrichment duration by 50%. The enriched samples are then transferred onto nitrocellulose membranes on brucella plates. To improve the sensitivity and specificity of the method, 0.45 &#181;m and 0.65 &#181;m pore-size filter membranes were evaluated. Data revealed a 29-fold increase in cell recovery with the 0.65 &#181;m pore-size filter compared to the 0.45 &#181;m pore-size without impacting specificity. The highly motile characteristics of Campylobacter allow cells to actively move through the membrane filters towards the agar medium, which enables effective isolation of pure Campylobacter colonies. The protocol incorporates multiplex quantitative real-time polymerase chain reaction (mqPCR) assay to identify the isolates at the species level. This molecular technique offers a reliable and efficient means of species identification. Investigations conducted over the past twelve years involving retail meats have demonstrated the ability of this method to enhance recovery of Campylobacter from naturally contaminated meat samples compared to current reference methods. Furthermore, this protocol boasts reduced preparation and processing time. As a result, it presents a promising alternative for the efficient recovery of Campylobacter from meat. Moreover, this procedure can be seamlessly integrated with DNA-based methods, facilitating rapid screening of positive samples alongside comprehensive whole-genome sequencing analysis.

Campylobacter is the leading cause of bacterial foodborne gastroenteritis worldwide. Despite establishments enacting measures to reduce the prevalence throughout their facilities, contaminated products consistently reach consumers. The technique developed over the past twelve years addresses limitations of existing methods for isolating and detecting Campylobacter spp. from raw meat.

This article presents a rapid yet robust protocol for isolating Campylobacter spp. from raw meats, specifically focusing on Campylobacter jejuni and ...