BACTERIAToxicology

Team 2

Benjamín Miranda Pineda A01181275Francisco Nava A00756599Stefano Spatafora Salazar A01190651

IntroductionMost bacteria are harmless, nevertheless some of them result to be pathogenic.

Mechanisms in which bacteria cause disease- Nutrients (Iron capture with siderophores)

- Direct damage- Toxin production (Endotoxins and exotoxins)

Objective

To show the most common types of pathogenic bacteria that might be present in the industrial environment, putting in risk the life and health

of customers.

Bacteria

● E. coli● Salmonella● Staphylococcus aureus● Clostridium perfringens

E. coli Introduction/Importance

● E. coli O157:H7 is a foodborne pathogen● Principal cause of foodborne illness worldwide.● Is the most common serotype of

enterohemorrhagic E. coli (EHEC).● Ground beef is an important raw agricultural

commodity and the major vehicle for transmission● International concern - Prevalence in meat ranging 0.1 %-5% and

prevalence in cattle ranging from 1.5%-28% ● The number of hospitalizations and deaths due to foodborne

transmission of this pathogen was estimated to be 1,843 and 52 per year

E. coli Hazard Identification - Biology of the pathogen

● Gram-negative, facultatively anaerobic, rod-shaped bacteria.● Commonly found in the lower part of the intestine of warm-blooded

animals.● E. coli 0157:H7 is a particular serotype, acid-tolerant, capable of

surviving in acid foods and during passage through the stomach.● Produces three toxins which stimulate intestinal secretion.● The infectious dose is about 10-100 cells and the vehicle of infection

include undercooked meat, post-pasteurized contaminated dairy products, apple juice and direct animal contact.

● Also known as Shiga-toxin producing E. coli (STEC). Shiga toxin is composed of two major subunits (A and B)

E. coli Hazard Characterization - Relationship with health

● Infection can result in moderate tosevere disease or death (childrenunder five years and the elderly).

● Incubation period: three to nine days.● The illness lasts 2-9 days; however with complications it may last for

months (permanent damage or even death)● Causes severe hemorrhagic colitis. 10% patients can develop HUS.

Long-term renal dysfunction occurs in about 10-30% of survivors of HUS.● Toxin damage to the brain leads to seizures, coma and possibly death.

E. coli Hazard Characterization - Relationship with health

Sympthoms:

- Hemorrhagic colitis- grossly bloody diarrhea- severe abdominal pain- Vomiting, but no fever- Hemolytic uremic syndrome (HUS): prodrome of

bloody diarrhea, acute nephropathy, seizures, coma, and death- The elderly may develop thrombotic thrombocytopenic purpura: similar

to HUS but also fever and central nervous system disorder

E. coli Sources of contamination

● Cattle are a major reservoir of EHEC.● Surfaces of beef carcasses during the slaughtering.● The process of trimming and subsequent grinding

distributes the pathogen throughout the ground meat.

Risk scenario: under cooking survival of the pathogen infection.● Cross-contamination in kitchens and food service establishments.● Other foods (lettuce, sprouts, fruit juices, vegetables, raw milk) and water.● Person-to-person (day care centers).● Direct contact with animals carrying the organism is also a recognized source

of infection

E. coli Toxicological effect mechanism

● Toxins (VTl and VT2) are similar to Shigella dysenteriae toxins.● Destroy the intestinal cells of the human colon.● The intestinal wall is perforated (turns bloody).● Also known as Shiga-toxin producing E. coli

(STEC). Shiga toxin is composed of two majorsubunits (A and B).

● Currently no treatment is available for EHECinfections.

● The use of conventional antibiotics exacerbatesShiga toxin-mediated cytotoxicity (higher risk ofdeveloping HUS).

E. coli Lethal doses

● It is difficult to establish a relationship level of E. coli 0157:H7 consumed and the probability of illness.

● Fewer than 100 cells, to as low as a dozen or so cells, can cause illness.● To establish a food safety objective (FSO) for this example, it is important for

the value to be necessarily conservative to reflect the degree of uncertainty, the relatively low infective dose, and the severity of the illness.

● The concentration of E. coli0157:H7 in raw ground beef shall not exceed 1 cfu 250 g-l.

● The FSO is equivalent to no greater than 1 cell per two patties with each weighing 125 g, a common weight for commercially manufactured ground beef patties

E. coli Legislation (maximum allowed levels)

European Community Council

Meat preparations: Council Directive 88/657/EEC laying down the requirements for the production of, and trade in, minced meat, meat in pieces of less than 100 g and meat preparations and amending Directives 64/433/EEC, 711118/EEC and 72/462/EEC.

E. coli Legislation (maximum allowed levels)

Milk and milk products: Council Directive 92/46/EEC laying down the health rules for the production and placing on the market of raw milk, heat treated milk and milk-based products.

E. coli Legislation (maximum allowed levels)

Egg products legislation: Legislation dealing with egg products is to be found in Council Directive 89/437/EEC. This covers products only, made from the eggs of either chickens, ducks, geese, turkeys, guinea-fowls or quails.

● The principal bacteria employed as indicators are the coliforms. HOWEVER traditional methods are prolonged and tedious.● Rapid methods aim to:

○ (1) replace the enrichment step (which requires a prolonged growth period) with a concentration step, i.e. immunomagnetic separation or

○ (2) to replace the end-detection method (usually colony development with prolonged incubation period), i.e. impedance microbiology and bioluminescence

● Current methods require approximately 10**5 organisms per ml for reliable detection. Since the regulatory requirement is the ability to detect 1 cell in 25 g of food a concentration factor of 10**7 is necessary.

●

Separation and concentration techniques

Immunomagnetic separation. The total time required for sampling and detection was reduced by one day due to the replacement of the selective enrichment step (overnight incubation) with the immunomagnetic separation (10 min). IMS is recognized as the most sensitive method for E. coli 0157: H7 and has been approved in Germany and Japan.

Detection techniques

ELISA. The technique generally requires the target organism to be 10**6 cfu/ml. Sensitive to 1-2 mg per ml culture filtrate. A positive result is obtained within 20 min and has a sensitivity limit of 104 cfu/ml.

ATP bioluminiscence. Since the molecule adenosine triphosphate (ATP) is found in all living cells (eucaryotic and procaryotic) its detection is indicative of living material being present. Sensitive to 1-2 mg per ml culture filtrate

E. coli Analytical techniques

Salmonella Chemical characteristics

There are two types of Salmonella as pathogens Typhoidal and non-Typhoidal.Unlike many other types of bacteria which produce toxic compounds, Salmonella is toxic by itself.

- Toxicity is associated with Lipid A, and immunogenicity with LPS from the cell wall.

- LPS are complex amphiphilic molecules with a mw of about 10 kDa.

Salmonella Health relationship

The physiological activities of LPS are mainly mediated by Lipid A components of LPS.

Powerful biological response that stimulate mammalian immune system.

Infection by Salmonella leads to Salmonellosis disease, which includes the development of:

- Diarrhea- Fever- Vomit- Abdominal cramps

Salmonella Sources of contamination

According to the Mayo Clinic, Salmonella is a bacteria that is transmitted through human or fecal contamination of food.

Most common ways of transmission and sources:- Foods of animal origin- Improper handwashing- Improper cleaning food preps areas- Pets

At the industry level, chicken eggs have been a wide example of Salmonella industrial risks of contamination. Nevertheless any type of food might represent a transmission way.

Salmonella Health effects

In acute infections, Salmonella can develope Salmonellosis and Typhoid fever or even Sepsis.

SalmonellosisRegular infection on the digestive system that provoke symptoms mentioned before.

Typhoid feverTyphoid fever occurs when Salmonella enters the lymphatic system and develop a systemic form of salmonellosis.

SepsisOccurs when a overreaction of the inmune system is achieved. This reaction can lead to death.

Salmonella Doses and Legislation

The severity of illness depends on the quantity of bacteria ingested.

Standards (National)No present in 25 g sample (NOM-242-SSA1-2009)

Standards (International)No present in 25 g sample (Australia & new Zealand FSA)

Salmonella Analytical techniques

In Mexico, the norm (NOM 114 - SSA1 -1994) is followed when trying to determine the presence of Salmonella in food products.

- Cultivo- Selección y aislamiento- Identificación- Prueba de Ureasa- Confirmación serológica- Informe

Staphylococcus aureus● Gram-positive● Non-sporing bacteria● Produces enterotoxins (SEs)

○ Staphylococcal food poisoning (SFP)

○ Symptoms: Nausea, violent vomiting, abdominal cramps, diarrhea

○ Rapid onset (2-8 hrs. after ingestion)

○ Wear off in 24 hours

S. aureus enterotoxins (SEs)● Exoproteins

○ Production → between exp. and stationary phase● 22 - 28 kDa● Heat-resistant● Growth at low aw● Active at low pH

○ Active when S. aureus destroyed● Resistant to proteolytic enzymes● Possess antigenic activity

SEs: Mode of action

● Penetrate the gut lining → activate immune responses

● Inflammatory mediators (histamine) → causes vomiting

● Inflammation of stomach and upper small intestine○ inhibition of water and electrolyte reabsorption →

diarrhea● Carboxymethylation of histidines (His61) → non-

enterotoxic

SEs: Doses and Legislation

Severity of illness depends on amount of food ingestedInfectious dose: 20-100 ng of enterotoxin

LD50: 20 μg/kg bw (monkeys, intravenous route).

Legislations:National:<1000 cfu/g in seafood (NOM-242-SSA1-2009)None in food for infants (NOM-131-SSA1-1995)

International:<100 cfu/g in ready-to-eat foods (NSW Food Authority, UK Food Protection Agency)

S. aureus: Sources of contamination and Analytical techniques

● Main foods: Meat and dairy products○ 7.2 - 60°C

● Food handlers○ Manual contact○ Respiratory secretions

● Food Processing○ Biofilms

Analytical Techniques:Sandwich ELISA:-Rapid (4hr.)

-Sensitive (0.0282 ng/ml)

-High specificity

Coagulase biochemical test (NOM-115-SSA1-1994)

Clostridium perfringens● Gram-positive● Anaerobic bacteria● Forms heat-resistant spores● C. perfringens enterotoxin (CPE)● Causes food poisoning

○ Abdominal cramps and diarrhea○ 8-12 hrs after ingestion

● Produced during sporulation

CPE● 35 kDa polypeptide (319 residues)● Two functional domains:

○ N-terminal region → mediates toxic effect

○ C-terminal region → receptor-binding region

○ C-terminal by itself is non-toxic● Released at the completion of sporulation

CPE: Mode of action

● C-terminal binds to claudins (receptors)○ At the tight junctions between

the epithelial cells of the gut wall

● N-terminal forms pores in membrane of enterocytes○ Alters permeability○ Cell death○ Inhibits water absorption

CPE: Doses and Legislation

Severity of illness depends on amount of food ingested

LD50: 81 μg/kg bw (mice, intravenous route).

Legislations:International:<100 cfu/g in meat products (RTCA 67.04.50:08 Reglamento Técnico Centroamericano)

C. perfringens: Sources of contamination and Analytical techniques

● Naturally found in human intestines● Food handling

○ raw materials, cooking, slow cooling● Main foods: Meat products

○ 10 - 54°C

Analytical Techniques:Sandwich ELISA:-Highly sensitive (1 ng/ml)-Highly specific (differentiates enterotoxin-positive from enterotoxin-negative strains).

OutbreakThe 1985 California Listeria Cheese Outbreak

The 1993 E. coli Jack in the box outbreak

- Mexican Cheese from Jalisco company- Deadliest foodborne illness outbreak in the U.S. - Pasteurized milk might have been mixed with

unpasteurized milk by the technician.- 142 cases of Listeriosis, more than 50 people died.

- Hamburger beef in discount Monster Burger “So good is scary”- The beef was not cooked long enough to kill E. coli.- 742 cases of E. coli 0157:H7 infections were detected.- 5 people died.

Containment

SUPERBUG IN INDIA

https://www.youtube.com/watch?v=uu4OC4eEIL8

References● Argudín, M., Mendoza, M., & Rodicio, M. (2010). Food Poisoning And Staphylococcus Aureus Enterotoxins.Toxins, 2, 1751-1773.● Briggs, D., Naylor, C., Smedley, J., Lukoyanova, N., Robertson, S., Moss, D., ... Basak, A. (2011). Structure of the Food-Poisoning

Clostridium perfringens Enterotoxin Reveals Similarity to the Aerolysin-Like Pore-Forming Toxins. Journal of Molecular Biology,413(1), 138-149.

● Cavalcanti, M., Porto, T., Porto, A., Brandi, I., Filho, J., & Junior, A. (2004). Large scale purification of Clostridium perfringens toxins: A review. Revista Brasileira De Ciências Farmacêuticas, 40(2), 151-164.

● Dinges, M., Orwin, P., & Schlievert, P. (2000). Exotoxins Of Staphylococcus Aureus. Clinical Microbiology Reviews, 13, 16-34.● FDA. (n.d.). BBB - Staphylococcus aureus. Retrieved February 20, 2015, from http://www.fda.

gov/Food/FoodborneIllnessContaminants/CausesOfIllnessBadBugBook/ucm070015.htm● FDA. (2011, January 1). BAM: Staphylococcal Enterotoxins. Retrieved February 21, 2015, from http://www.fda.

gov/Food/FoodScienceResearch/LaboratoryMethods/ucm073674.htm● Gill, M. (1982). Bacterial Toxins: A Table of Lethal Amounts. Microbiological Reviews,46, 86-94.● Gutierrez, D., Delgado, S., Vazquez-Sanchez, D., Martinez, B., Cabo, M., Rodriguez, A., ... Garcia, P. (2012). Incidence of

Staphylococcus aureus and Analysis of Associated Bacterial Communities on Food Industry Surfaces. Applied and Environmental Microbiology, 78, 8547-8554.

● Kuang, H., Wang, W., Xu, L., Ma, W., Liu, L., Wang, L., & Xu, C. (2013). Monoclonal Antibody-Based Sandwich ELISA for the Detection of Staphylococcal Enterotoxin A.International Journal of Environmental Research and Public Health, 10, 1598-1608.

● Lindström, M., Heikinheimo, A., Lahti, P., & Korkeala, H. (2011). Novel insights into the epidemiology of Clostridium perfringens type A food poisoning. Food Microbiology,28(2), 192-198.

● McClane, B., & Strousse, R. (1984). Rapid detection of Clostridium perfringens type A enterotoxin by enzyme-linked immunosorbent assay. Journal of Clinical Microbiology, 19(2), 112-115.

References● Mcclane, B. (2001). The complex interactions between Clostridium perfringens enterotoxin and epithelial tight junctions. Toxicon,

39(11), 1781-1791.● NSW Food Authority. (2009, January 1). Microbiological quality guide for ready-to-eat-foods. Retrieved February 22, 2015, from

http://www.foodauthority.nsw.gov.au/_Documents/science/microbiological_quality_guide_for_RTE_food.pdf● Reglamento Técnico Centroamericano. (2009, January 1). ALIMENTOS. CRITERIOS MICROBIOLÓGICOS PARA LA INOCUIDAD DE

ALIMENTOS. Retrieved February 19, 2015, from http://www.cacia.org/documentos/Criterios_microbiologicos.pd.pdf● Schelin, J., Wallin-Carlquist, N., Cohn, M., Lindqvist, R., & Barker, G. (2011). The formation of Staphylococcus aureus enterotoxin

in food environments and advances in risk assessment. Virulence,2(6), 580-592.● Secretaría de Salud. (1994, January 1). NORMA OFICIAL MEXICANA NOM-115-SSA1-1994, BIENES Y SERVICIOS. MÉTODO PARA

LA DETERMINACIÓN DE STAPHYLOCOCCUS AUREUS EN ALIMENTOS. Retrieved February 22, 2015, from http://www.salud.gob.mx/unidades/cdi/nom/115ssa14.html

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