IV MICROBIAL FOODBORNE PATHOGENS

A. MICROBIAL TOXINS

Definition

Given the proper dose and route of administration, any substance can be toxigenic. It is important to realize the limitations of a definition. The term 'microbial toxin' has been defined as a proteinaceous substance that is a specific product of the metabolic activity of a living organism and is notably toxic (causes or contributes to an unhealthy state) and capable of inducing antibody formation when injected into the appropriate host. This definition excludes low molecular weight toxins of the fungal class and non-protein toxins such as 'endotoxin'. It is clear that any definition of the word toxin will be limiting.

For the purpose of this discussion, toxins will be defined as:

Any substance produced by an organism and when introduced into a susceptible host in a "normal" dose is capable of producing or contributing to an unhealthy state in that host.

Kinds of Microbial Toxins

1. Mycotoxins

A. Aflatoxins

Produced by Aspergillus flavus

Aflatoxins are secondary metabolites. Zinc increases the production while copper decreases the production of toxin. The toxin is produced in the fungal mycelium and is subsequently released into the substrate. Aflatoxin is usually introduced into the host by way of food. FDA has set of level of 15 ug/kg as the maximum allowable limit in peanuts.

Aflatoxins can cause acute or chronic effects depending on the dose. These effects can be toxigenic, mutagenic, teratogenic or carcinogenic.

Ducklings or rainbow trout are typically used as biological models to study and detect aflatoxins. Aflatoxins consist of 13 subclasses, four being the most common: B1 B2 G1 G2. The letters correspond to the fluorescent color of the compound while the numbers denote the mobility (Rf) of the compound via thin layer chromatography. Aflatoxin B1 is the most toxic and the most common. LD50 for oral challenge in ducklings is 0.36 mg/kg. Acute intoxication due to aflatoxin is characterized by hemorrhage in tissues (usually the liver). It also inhibits DNA synthesis by inhibiting RNA polymerase. Tumor formation can also occur in the liver.

Principle Toxic Effects of Some Aspergilli Mycotoxins

Other Fungi That Can Produce Mycotoxins

Alternaria Penicillium
Aspergillus Pithomyces chartrum
Chaetomium Rhizopus
Cladosporium Paecilomyces varioti
Mucor hiemalis Fusarium
Sclerotina sclerotiorum Stachybotrys atra
Tricoderma lignorum

Principle toxic effects of some mycotoxins produced by fusaria

2. BACTERIAL TOXINS

Bacterial toxins have been classified as either endotoxins or exotoxins. This nomenclature has been replaced by more descriptive terms.

Endotoxin usually refers to the lipopolysacchride (LPS) portion of the gram negative cell wall. The term exotoxin is rarely used today. Many of the classical exotoxins are actually contained inside the bacterial cell and released only upon cell autolysis.

A more appropriate classification of bacterial toxins is based on the mode of action of the toxin moiety, location of mode of action or tradition.

a. Neurotoxins

i. Tetanus Toxin

organism - Clostridium tetani

production - Spores of this organism gain entry into the host usually by puncture wound. Germination and growth occurs in reduced atmosphere or with the metabolic activity of facultative anaerobic bacteria. Toxin is released from the bacterial cell through autolysis or chemotherapy as a single peptide of molecular weight ca. 150K. The toxin binds to gangliosides at the synaptic junction and travels to the anterior horn of the spinal cord through the axon ( intra axonal retrograde transport).

pathology - Tetanus toxin is composed of two subunits "A" and "B". The mode of action seems to be one of blocking feedback inhibition of motor neuron transmission. Toxin blocks the release of glycine at the interneuron motor neuron synaptic junction resulting in spastic paralysis. Rare but not unheard of is the ability of the toxin to block acetyl choline release at the neuromuscular junction causing flaccid paralysis.

Damage occurs at the muscle and death usually results from respiratory failure.

ii. Botulinum Toxin

organism - Clostridium botulinum

production - Toxin entry is usually through ingestion of food. Spores present in food germinate producing toxin which is released as a single peptide. The toxin is absorbed through the gut and reaches the nervous system via the lymphatic system.

pathology - Botulinum toxin is composed of two subunits "A" and "B" of ca. 150K. Seven types are recognized:

A. most potent, food
B. food

C. fowl

D. cattle
E. fish
F. phage

        E. phage

At the nerves, the toxin attaches to the pre-synaptic terminals of the cholinergic nerves resulting in paralysis. Twelve-24 hours is required for symptoms to appear after ingestion of the toxin.

Symptoms:

1. fever is absent, temperature is reduced

2. mental status, pulse is normal

3. decreased bowel sounds

4. constipation

5. blurred vision, dilated pupils

6. sore throat

Death is due to asphyxiation as long as 36 days after onset.

Sudden Infant Death Syndrome (SIDS) is caused by ingestion of spores from soil contaminated food or honey. Toxin production occurs in the gut.

b. Enzymatic Toxins

i. Cholera Toxin

organism - Vibrio cholerae

gram (-) serotype Ogawa, Inaba, O1; biotype El Tor

production - Cholera is due to ingestion of the organism via food or water. Greater than 10⁸ cells are required to survive the acidity of the stomach. The bacterium multiply in the small intestine adsorb to the lining without invasion. Most of the toxin is inside the bacterial cell but is released upon autolysis.

pathology - The toxin is composed of "A" (28K) and "B" (5-11K) subunits. The "B" unit binds to the GM1 (gaglioside) receptor of the host cell. Cells which lack this receptor are resistant to the toxin. Lateral fluidity of the GM1 in the membrane of the host cells allows binding of all 5 fragments. The "A" fragment is released inside the host cell in an unfolded state, refolds and binds to adenylate cyclase. This binding occurs at a protein of 42K. The host protein is ribosylated.

In a normal adenylate cyclase system, cAMP levels are regulated by hormones which stimulate the production of GTP which in turn stimulate the formation of cAMP. GTPase hydrolysis causes cAMP production to stop. Cholera toxin blocks this inhibition resulting in high cAMP levels and subsequent water accumulation.

Effective antibody treatment is towards the "B" subunit.

1. 20 liters/day water loss

2. no tissue damage

3. non-reversible - 24 hour for new villi to generate

 

ii. E. coli Enterotoxins

organism - Escherichia coli (ETEC)

production - Two types of toxin are produced by some E. coli. LT (heat labile) and ST (heat stable toxin) appear to be associated with the membrane of the bacterial cell. Cells are ingested along with food or water. The organism multiplies in the gut with subsequent toxin production. The toxin has been shown to be encoded by a plasmid. The native form of the toxin has been reported to be 80-105K protein. However, the toxic form may be much smaller suggesting that proteolytic cleavage is necessary for toxic effects. ST is non-antigenic with low molecular weight of 4-5K. ST appears to be more strongly bound to the bacterial cell membrane than LT.

pathology - ST stimulates increases in cyclic GMP rather than cyclic AMP. Research suggests that this toxin causes diarrhea in a similar manner as does increases of hormones.

c. Membrane Damaging Toxins

i. Perfringens Toxin

organism - Clostridium perfringens

production - Spores are ingested along with food. Spores germinate in the gut and infection follows. Toxin is elaborated in the gut as a structural part of the spore coat. Upon lysis of the cell the spore and the toxin are released. The toxin is a protein ca. 33-40K. Five types are recognized.

A

B

C

D

E

pathology - Tissue destruction, glucose uptake is inhibited. Diarrhea is due to loss of water uptake not secretion.

ii. Staphylococcal Enterotoxin

organism - Staphylococcus aureus

production - Toxin is produce and elaborated into the environment (food). Ingestion of the preformed toxin results in rapid symptoms of vomiting. Toxin is a protein of molecular weight of 28-35K which is heat stable. Eight types are recognized.

A

B

C1

C2

D

E

F         toxic shock

Exfoliatin SSSS

pathology - Less than 20 ng/kg body weight is toxic. Toxin does not primarily act on gut tissue. Toxin activates receptors of the abdominal viscera, stimulating the vomiting center via the vagus nerve. Thus the toxins can be considered a neurotoxin. Although diarrhea is sometimes present the toxin does not stimulate cAMP nor shut down glucose pump.

ii. Bacillus cereus Enterotoxins

organism - Bacillus cereus

production - Two distinct toxins are produced by this organism. Diarrhea type toxin and emetic type toxin. The diarrhea type is a protein of ca. 50K while the emetic type is small <5K and heat stable most likely associated with the spore coat. Both toxins may be ingested preformed.

pathology - The diarrhea type toxin appears to activate the adenylate cyclase system. The mechanisms of the emetic toxin are unknown.

iiii. Shigella Enterotoxin

organism - Shigella dysenteriae     E. coli O157:H7   EHEC

production - Unknown

pathology - The mechanisms of this toxin are unknown. Non-invassive toxigenic strains are nonpathogenic while nontoxigenic invasive strains are pathogenic. The toxins are thought to produce cytolytic effects partially responsible for the symptoms of the disease.

IV B. INTOXICATIONS

1. Staphylococcus aureus

First recognized in the 1880s.

Animal and humans are reservoir. with 30-50% of humans carry the organism in the throat, nose and skin.

Coagulase positive

Staphylococcus aureus

S. intermidius

S. hyicus

Coagulase negative

S. epidermidis

Methodology

Plate primary homogenate onto Baird-Parker Agar 1ml over 3 plates incubated 48 h 37oC. Count black colonies with or without opaque zone &/or clear halo extending beyond opaque zone.

Transfer 5 typical colonies to BHI broth (2ml) incubated 24 h 37oC. 0.5ml of culture is added to 0.5 ml coagulase plasma with EDTA in a 10x75 culture tube. Incubate 37oC for up to 24 h for clot formation.

Transfer 0.2 ml of boiled BHI culture to wells cut in DNA agar plates. Incubate 4 h at 37oC or 2 h at 50oC. Pink halos are indicative of positive TNase activity.

Halophilic - may grow upto 20% NaCl.

Osmotolerant - may grow at Aw values 0.83.

Poor competitor

FOODS

meat

milk

pastry

ice cream

dried milk

hard boiled eggs

GENETICS OF TOXIN PRODUCTION

Tox gene for SEA & SEB is chromosomal

SYMPTOMS

Vomiting

Diarrhea

1-6 hours after ingestion

Dose <1 ug of SEA (144 ng)

IV B. INTOXICATIONS

2. Clostridium botulinum

gram +, anaerobic, rod-shaped, spore-forming bacterium which produces a neurotoxin.

Types

A man USA, vegetables, proteolytic

B man Europe, meat

E man psychrotrophic, fish, non-proteolytic

C animals

D animals

F 3 cases

Inhibitory pH 4.6

Inhibitory NaCl 10%

Minimal Aw 0.94
D100 spores 25 min

Z 10 C

12D 2.4 min @ 121oC

toxin inactivation 5 min @ 85oC

SYMPTOMS

Incubation hours - days (12-36 h)

vision

mouth

fatigue

respiratory

death

IV B. INTOXICATIONS

3. Bacillus cereus

First reported in the early 1900 and characterized in the 1950s.

Large gram positive rods(1 x 5 um), motile, sporeforming rod (central not swollen).

Spores grow aerobically while vegetative cells are facultatively anaerobic.

D100 3 min

DISEASE:

"diarrheal syndrome" 8-16 h incubation

cramps, watery diarrhea.

intoxication

associated with hemolysin

50,000 MW protein

increases cylic-AMP

"emetic syndrome" 1-5 h onset

vomiting.

intoxication

<10,000 MW protein?, heat stable

METHODS:

Mannitol Egg Yolk Polymyxin Agar (MYP)

lecithin hydrolysis positive, mannitol negative

IV C. INFECTIONS & TOXICOINFECTIONS

1. Vibrio cholera

Common infectious agent in India through 1817. China 1820, Japan 1822, Europe 1823, North America 1832. Koch in 1883 showed that cholera was caused by an infectious agent.

Currently we are experiencing the 7th pandemic of cholera.

Gram negative, polar flagellated, oxidase & catalase positive, ferment glucose with no gas, facultatively anaerobic, curved rod.

Genus is comprised of 28 species with 10 able to produce disease.

Vibrio cholerae is classified in two groups:

O1 (toxin producing)

Classic El Tor

Inaba

Ogawa

Hikojima

non O1 (non toxigenic)

SYMPTOMS:

incubation 5-6 days

cholera gravis - pass >1 liter/hour.

10% of infected require hospitalization

2% infected with El Tor are severe

75% are asymptomatic.

Tetracycline (500mg orally every 6 h for 3 days)

METHODOLOGY:

25 g enriched for 6-8 hours at 35oC in alkaline peptone water (pH 8.4) and gelatine phosphate saline.

streak to thiosulfate citrate bile salts agar (TCBS) incubated at 35oC for 24 h. Pick yellow or green colonies for biochemical and virulence testing.

2. Vibrio vulnificus

Extremely infectious, invasive bacterial pathogen.

Causes both intestinal and wound infections.

Halophilic (1-3%), lactose positive, survives poorly at 4oC.

Found in sea water, 85% of cases involve consumption of raw oysters.

METHODOLOGY:

isolation on TCBS agar

PATHOLOGY

Toxin 56,000 MW exhibiting cytolytic activity.

Iron present in serum increases virulence.



3. Vibrio parahaemolyticus

Recognized as food pathogen in 1950 Japan.

Obligately halophilic, sea food related.

Wagatsuma agar containing human blood demonstrates pathogenic strains known as Kanagawa hemolysin positive.

Virulence is unknown.

4. Clostridium perfringens

First associated with food poisoning in the 1940s

Type A is associated with food

Optimal growth is 43-45oC generation time 7.1 min.

Toxin is formed during sporulation.

Toxin may be transferred via plasmid.

Symptoms

8-24 h after ingestion

diarrhea and cramps

Found in soil, poultry 50%, feces.

Methodology

Tryptose-sulfite-cycloserine agar

incubated 37oC anaerobically

stormy fermentation - milk with iron at 45oC

5. SALMONELLA

Salmonella, coined by Lignieres in 1900 in honor of D.E. Salmon.

More than 2300 species are recognized, all potentially pathogenic.

All salmonellae are characterized based on somatic (O) and flagellar (H) antigens.

Five subgenera were classified by Kauffman and named by Ewing:

I S. enterica associated with warm blooded animals

II S. salamae

IIIa. S. arizonae associated with cold blooded animals

IIIb. S. diarizonae diphasic species of III

IV. S. houtenae

V. S. bongori

VI. S. indica

Temperature

growth 3-47oC

Salt

growth is usually inhibited by 3-4% NaCl.

PH

optimal growth is 6.5 - 7.5 (4.5 - 9.0)

Pathogenesis

symptoms of infection

diarrhea

cramps

fever

nausea

vomiting

prostration

septicemia

onset 8-72 hours

treatment

chloramphenicol, ampicillin or trimethoprim-sulfamethoxazole

Infective dose

<50 cells of S. napoli in chocolate bars manufactured in northern Italy resulted in clinical symptoms.


FDA Culture Method

A. Preenrichment

1. Objective

2. Sample Size
3. Media

4. Incubation

B. Selective Enrichment

1. Objective

2. Sample Size

3. Media tetrathionate selenite cystine

4. Incubation Conditions

C. Selective Isolation and Differentiation

1. Plating Media and Incubation Conditions

a. Xylose Lysine Desoxycholate Agar (XLD)

red with black center

sodium thiosulfate - ferric ammonium citrate

lactose, sucrose, xylose

b. Hektoen Enteric Agar (HE)

blue - green with black center

sodium thiosulfate - ferric ammonium citrate

salicin, sucrose

c. Bismuth Sulfite Agar (BS)

brown - black

glucose

D. Screening Isolates

1. Media and Incubation Conditions

a. Triple Sugar Iron Agar (TSI)

red slant - yellow butt - black

lactose, sucrose, glucose

b. Lysine Iron Agar (LIA)

purple - black

glucose, lysine

95% lactose (-)

5% lactose (+)

98% H2S (+)

2% H2S (-)

99% lysine (+)

1% lysine (-)

E. Serological Confirmation

a. Somatic "O" slide agglutination

b. Flagella "H" tube agglutination

6. Enteropathogenic Escherichia coli

First described by Dr. Escherich in 1885.

Biotype 1 IMViC ++-- 95%

Biotype 2 IMViC -+-- 5%

Categories

1. enteropathogenic (EPEC)

2. enteroinvassive (EIEC)

3. enterotoxigenic (ETEC)

4. enterohemorrhagic (EHEC) or O157:H7

1. EPEC

1954 Bray & Neter - summer diarrhea or infantile diarrhea

serogroups O18ab, O18ac, O26, O44, O55, O86, O111, O114, O119, O125, O126, O127, O128ab, O142, O158.

Symptoms

diarrhea, fever, vomiting, cramps

duration 6 hours - 3 days (24 h)

onset 17 h - 72 h (36 h)

2. EIEC

first described in 1943

often non-motile, anaerogenic, lactose negative

serogroups O28ac, O29, O124, O136, O143, O144, O152, O164, O167

similar to Shigella

Symptoms

chills, fever, headache, muscle pain, cramps, diarrhea or dysentery

onset 8-24 h (11h)

Sereny Test

Tissue culture

EIA

Gene Probe

3. ETEC

first described in 1968 'travelers diarrhea'

all E. coli producing heat labile toxin (LT) or heat stable toxin (ST).

serogroups O6, O8, O15, O20, O25, O27, O63, O78, O80, O85, O115, O128ac, O139, O148, O153, O159, O167.

Symptoms

watery diarrhea (rice water), fever, cramps, nausea

duration 3-9 days

onset 8-44 h (26h)

rabbit ligated ileal loop assay

vascular permeability factor

infant mice

sealed mouse model

tissue culture

EIA

Gene Probes

4. EHEC

Identified in 1982 as a pathogen.

93% of E. coli are sorbitol (+), O157:H7 is sorbitol (-).

Most E. coli are glucuronidase (+), O157:H7 is (-).

(MUG 4-methyl-umbelliferone glucuronide)

does not grow at 45.5oC

Symptoms

hemorrhagic colitis - sudden onset of cramps followed by watery diarrhea followed by bloody diarrhea. Incubation is 3-9 days (4 days) duration 2-9 days (4 days). No fever.

hemolytic uremic syndrome (HUS) - acute renal failure bloody diarrhea, seizures, prolonged coma and death.

thrombotic thrombocytopenic purpura (TTP) - central nervous system involvement with blood clots in the brain, death.

1982 - Fast food chain 47 cases

Verotoxin (VT1), (VT2), gene is carried by phage.

VT3 -

7. Shigella

First described in late 19th century

S. dysenteriae serogroup A 2%

S. flexneri serogroup B 31%

S. boydii serogroup C 3%

S. sonnei serogroup D 65%

genetically similar to E. coli

non-motile, lactose (-), delicate

cases declined during the 1940s but have gradually increased during the past 30 years.

Bacillary dysentary - blood in diarrheal stool.

Symptoms

fever >105oF, convulsions 75%, headaches, Reiter's syndrome, HUS

incubation - 1-7 days persisting for 2 weeks.

As few as 10 cells may cause disease.

estimates of 300,000 cases per year in U.S.

Pathology

invasive, plasmid, shiga toxin

Methods

Enrichment

25 g mixed with 225ml Shigella broth (dextrose 0.1%, NaCl 0.5%, tryptone 2.0%, Tween 80 0.15%, buffer, novobiocin 3 ug/ml).

incubation at 42oC 20 h anaerobically.

streak to MacConkey agar incubate 20 h 35oC.

pick pink-translucent colonies to:

glucose + no gas

TSI R/Y-

lysine broth -

motility agar -

tryptone +/-

confirm serologically

8. Listeria monocytogenes

First described in 1926 by Murray et al.

psychrotrophic, halophilic, catalase (+), flagellated, small gram (+) rod, slightly hemolytic on blood agar.

Only L. monocytogenes is considered pathogenic.

Symptoms

onset 1 day to several weeks

flulike to fever, malaise, nausea, vomiting, pain, diarrhea.

imunocompromised individuals - septicemia death.

mortality rate as high as 30%.

Outbreaks

1981 cole slaw 34 cases of prenatal listeriosis and 7 cases of adult disease, fatality rate 27%.

1983 pasteurized 2% milk 7 cases were fetus and 42 cases of adult.

1985 soft cheese 86 cases of mother infant.

Methods

Preenrichment

25 g mixed with 225ml UVM (esculin, nalidixic acid, acriflavin).

incubation at 30oC 24 h.

transfer 0.1 ml to Fraser's broth incubate 24 h 35oC.

streak black tubes to MOX (moxalactam) agar incubate 48 h at 35oC.

pick 5 black colonies to TSA-YE plates incubate 24 h 35oC.

inoculate biochemical and CAMP blood agar for confirmation.

9. Yersinia enterocolitica

First described in 1939 in New York.

psychrotrophic, alkaline resistant, , flagellated, gram (-) rod.

serovars O3, O9, O5, O8.

Symptoms

fever, pseudoappendicitis, pain,.

gastroenteritis in young children.

1976 - 18 appendectomies

1982 - largest outbreak in world was in U.S. - milk

pig tongues

Pathology

plasmid, calcium, toxin?

10. Campylobacter jejuni

First described as Vibrio fetus in 1913.

Now considered the leading cause of acute bacterial gastroenteritis in humans

gram negative, curved rod, slightly thermophilic, microaerophilic, motile

species C. jejuni, C. fetus, C. coli, C. laridis (naladixic acid resistant)

Symptoms

incubation 2-5 days

duration up to 10 days or longer if untreated

fever >40oC, malaise, cramps, headache, diarrhea 8/day, dysentery

Pathology

as few as 500 cells may cause illness, invasion, toxin antigenically related to cholera and E. coli LT, erythromycin treatment

Growth and Survival

growth range >30oC to 45oC (optimal 42-43oC)

organism is 10 times more sensitive to thermal inactivation than Salmonella.

freezing reduces numbers 100 fold.

minimum pH for growth 4.9

growth in NaCl up to 1.5%

Epidemiology

unpasteurized milk 2500 school children

undercooked poultry military exercise

young adults birthday cake

Isolation and Detection

growth in reduced oxygen enrichment media

ferrous sulfate, metabisulfite, pyruvate

enrichment containing antibiotics

vancomycin, trimethoprim, polymyxin B, cycloheximide

plate on blood media

translucent, nonhemolytic, flat, gray-pink, small

phase contrast wet mount

thin, rapid corkscrew movement, comma shape

catalase positive, oxidase +, no fermentation

11. OTHERS

Aeromonas hydrophila, A. sobria

Facultatively anaerobic, gram negative, motile, catalase and oxidase positive, halophilic, psychrotrophic rods.

"cholera-like" diarrhea

"dysentery-like" diarrhea

toxin? "cholera-like, cytotoxin

invasiveness?, hemolytic?

Starch Ampicillin agar is used to enumerate organism.

Plesiomonas shigelloides

Facultatively anaerobic, gram negative, motile, catalase negative and oxidase positive, halophilic, psychrotrophic rods.

heat stable and heat labile toxins