Food Micro Flashcards
1
Q
BACILLUS CEREUS
A
- WAXY on blood agar
- No acid from mannitol
- Cells are 1-1.2 um diameter by 3-5 um in length containing endospores which are central or paracentral without swelling the vegetative cell
- Characteristics
- Gram positive rod (>9μm in diameter by 3-5μm in length)
- Vegetative or spore forming
- Motile
- Generally hemolytic
- Toxin producing (enterotoxin and emetic)
- Common soil saprophyte
- Grows well after cooking and cooling because heat treatment causes
- EPI
- It is ubiquitous in nature - naturally found in soil
- B. cereus spores can be found in dry products such as dry soups, dried dairy products, infant formula and seasoning
- Fried rice is a common cause of food poisoning due to long period between boiling the rice and consumption after frying (“Fried Rice Syndrome“)
- >10^5 organisms/gram needed to cause an illness
- Due to mildness of disease many cases go unreported
- Can be ingested and found in the human intestine as transitory micro-flora
- BACILLUS CEREUS FOOD POISONING
- Can produce 2 disease syndromes which are similar to Staphylococcus aureus and Clostridium perfringens syndromes
-
- Syndrome #1 - Emetic
- Onset time – 0.5 to 5 hrs
- Symptoms: nausea, vomitting, malaise and ocasionally diarrhea
- Implicated food – fried rice, cooked rice, pasta, corn meal dishes, porridge
- Toxin pre-formed (cereulide) within the food and heat stable
- Mechanism of toxin action: not well characterized, forms ion channels and holes in host cell membranes
- Infective dose is 10^5 to 10^7 organisms/gram
- Treatment – no specific treatment, self-limited illness
-
- Syndrome #2 - Diarrheal
- Onset time - 8-16 hrs (mean 12 hrs)
- Symptoms: abdominal pain, watery diarrhea and occasionally nausea
- Implicated foods - meat products, soups, vegetables, puddings, sauces, milk and milk products
- Toxin (enterotoxin) formed in the small intestine and is heat labile
- Mechanism of toxin action: pore formation and activation of adenylate cyclase enzymes resulting in intestinal fluid secretion
- The infective dose is 10^5 to 10^7 cells per gram of food
- Treatment - no specific treatment, self-limited illness
- Detection
- Optimal growth conditions: 30-37°C, but can grow at temperatures up to 55°C and in some cases down to 5°C
- pH: 4.3- 9.3
- Maximum NaCl % - 18
- Mannitol Egg Yolk Polymyxin B (MYP): standard media for plating B. cereus, but it has little selectivity so background flora is not inhibited and can mask the presence of B. cereus.
- Bacillus Cereus Rapid Agar (Bacara agar, AEX Chemunex): chromogenic selective and differential agar that promotes the growth and identification of B. cereus, but inhibits the growth of background flora.
- BACILLUS CEREUS ON EGG YOLK AGAR LECITHINASE POSITIVE
- BACILLUS CEREUS ON MYP AGAR PLATES SHOWING DIFFUSE LECITHINASE ACTIVITY
- Isolation of 10^5 B. cereus per gram from the epidemiologically implicated food
- Isolation of B. cereus from stools of 2 or more ill persons (symptomatic persons)
2
Q
Clostridium perfringens
A
- General characteristics
- Anaerobic
- Large, gram-positive rods
- Motile
- Decompose proteins or form toxins
- Some do both
- Natural habitat is soil or intestinal tract
- Saprophytes
- Includes bacteria that cause botulism, tetanus, gas gangrene, and pseudomembranous colitis
- General Morphology and Identification
-
Plating on Tryptose Sulphite Cycloserine (TSC) plates
- Black Colonies
-
Plating on Tryptose Sulphite Cycloserine (TSC) plates
- Typical organisms
* Spores have larger diameter than rods
* Spore is placed centrally, subterminally or terminally
* Mobile via peritrichous flagella- Culturing
- Anaerobic, with some species being aerotolerant
- growth in ambient air
- Use of blood-enriched plates
- Colony forms
- Produce large, raised colonies
- Colonies can spread on agar surfaces
- Zone of beta-hemolysis on blood agar
- Growth characteristics
- Saccharolytic or proteolytic classification based on sugar fermentation or protein digestion
- Able to ferment a variety of sugars and can digest proteins
- Antigenic characteristics
- Have some antigens, but have specific soluble antigens that allow for grouping
- Culturing
- Typical organisms
- Public Health Significance
- Clostridium perfringens are ubiquitous
- Mode of transmission: C. perfringens spores can contaminate high protein foods (e.g. meats, poultry, and stew)
- Foods implicated: warmed over high protein foods (beef stews, pot pies, poultry, gravies, sauces, soups, refried beans)
- Clostridium illnesses
- Histotoxic group — tissue infections
- C. perfringens (type A)
- Enterotoxigenic group — gastrointestinal disease
- C. perfringens (type A)
- necrotizing enteritis (beta toxin-producing C.perfringens type C)
- Tetanus — C. tetani neurotoxin
- Botulism — C. botulinum neurotoxin
- Histotoxic group — tissue infections
- Onset Time: 6-24 hours
- Symptoms: Acute diarrhea, abdominal pain, And nausea. Vomiting and fever are uncommon
- C. perfringens Morphology
- large, rectangular bacilli
- gram-positive
- Spores: rarely seen in vitro or in clinical specimens (ovoid, subterminal)
- rapid spreading growth on blood agar mimics the growth of motile organisms
- Some strains are aerotolerant, especially on media supplemented with blood
- grow at a temperature of 20-50°C (optimum 45°C) and
- pH of 5.5-8.0
- Food Related to
- Illness associated with undercooked meats
- Laboratory tests for C. perfringens
- Reverse CAMP
- Microscopy
- Gram stain
- Capsules
- gas from glucose fermentation
- in vivo toxicity testing and identification of the specific toxin types involved
- Blood agar plates
- double zone of hemolysis on blood agar
- theta toxin and alpha toxin
- Nagler reaction
- precipitation in serum or egg yolk media
- Alpha toxin is a lecithinase
- Litmus milk
- “stormy” fermentation
- coagulation of milk due to large amounts of acid and gas from lactose
- ID = 10^8 vegetative cells needed to cause symptoms
- Incubation time to Illness: 8 – 24 h
- Severe diarrhea, shivering, headaches, severe abdominal pain, dehydration
- No fever
- Plating on Tryptose Sulphite Cycloserine (TSC) plates
- Incubate anaerobically
- Black colored colonies
- Most probable Numbers (MPN) in cooked meat or liver medium followed by streaking on neomycin blood agar
- ELISA – Toxin Detection Kits
- Can detect epsilon toxin with biological fluids
- Intestinal, peritoneal, pericardial
- PCR – primer/probe sets, qPCR
- Toxin-antitoxin neutralization test
- C. perfringens Prevention and Control
- Disinfect with soap and water
- Health care workers
- Should follow standard safety precautions
- Vaccine available for animals
- Follow FDA/USDA guidelines for proper handling of food
3
Q
What are toxins?
A
- A poisonous substance produced by living cells or organisms
- Stable, nonvolatile, tasteless:
- Small molecules
- Peptides
- Proteins
- Interact with enzymes, cellular receptors
- Cleave essential protein residues enzymes important in DNA replication
- Ingested, inhaled or injected
4
Q
Cellular attack mechanisms
A
- A-B type toxins
- Active and Binding components
- Interfere with internal cell function
- Botulinum, shiga and Shiga-like toxins
- Receptor-targeted toxins
- Bind cellular receptors triggering damaging transmembrane signals
- Staphylococcal enterotoxins, Gram negative toxins
- Membrane-damaging toxins
- Damage cellular or plasma membrane – pores formed
- Staphylococcus (Panton-Valentine leucocidin) PVL cytolysins leukocyte destruction and tissue necrosis
5
Q
Toxins produced by bacteria
A
- Staphylococcal food poisoning
- Clostridium botulinum
- Bacillus cereus food poisoning
6
Q
Staphylococcus aureus
A
- Can produce enterotoxins that might not be destroyed by cooking
- Staphylococcal toxins are very resistant to heat.
- Enterotoxins retain some of their biological activity in milk after pasteurization or heating at 121°C for 28 min.
- Toxins are fast-acting, causing symptoms 1-7 hours
- Last few hours to a day
- Symptoms
- Nausea
- Stomach cramps
- Vomiting
- Diarrhea
- Dehydration
- Headaches
- Muscle cramps
- Changes in blood pressure and heart rate
7
Q
Staphylococcal enterotoxins
A
- Single-chain proteins (26 to 29 kDa)
- Resistant to proteolytic enzymes such as trypsin and pepsin (survive through digestive tract)
- 5 classical serotypes:
- SEA, SEB, SEC1,2,3, SED, SEE
- More recently SEF, SEG, SEH, SEI
- All have emetic activity
- Intoxication dose of SE is less than 1 microgram
- 100 ng SEA
- Reached typically when S. aureus population exceeds 100,000 organism/g food
- Detection
- Micro slide gel double diffusion- requires at least 30-60 mg enterotoxin per gram food
- Radioimmunoassay
- Latex agglutination
- Several EIA methods
- Enzyme-linked fluorescent immunoassay (ELFA)
8
Q
Bacillus cereus enterotoxin
A
- Diarrheal type- large molecular weight toxin
- Vomiting (emetic) type- cereulide, an ionophoric low-molecular weight
- dodecadepsipeptide that is pH-stable and heat and protease resistant
- Stable at 121C for 30 min, 4C for 60 days, pH range 2-11
- onset
- Diarrheal type - 6-15 hours after consumption
- Emetic type 0.5 – 6 hours after consumption
- symptoms
- Diarrheal type - watery diarrhea, abdominal cramps, pain (mimic C. perfringens food poisoning), nausea (vomiting is rare)
- Emetic type- nausea, vomiting similar to S. aureus foodborne intoxication
- detection
- Microslide gel double diffusion test to identify enterotoxin
9
Q
Clostridium botulinum
A
- General
- Anaerobic
- Gram-positive
- Spore-forming rod
- Produces a potent neurotoxin
- Spores are heat-resistant and can survive in foods that are incorrectly processed
- Botulinum toxin
- A, B, E and F cause human botulism
- C and D cause botulism in animals
- C and E can cause botulism in birds
- No outbreaks of type G reported
- Most strains produce only one type of toxin
- Foodborne botulism
- Severe type of food poisoning caused by ingestion of foods containing the toxin produced by C. botulinum
- Most often consumption of improperly processed or inadequately cooked home-preserved foods
- Home-canned or commercial-produced foods have been involved in outbreaks
- Almost any type of food that is not very acidic (pH above 4.6) can support growth and toxin production by C. botulinum.
- Salt concentration from 4% to 5% is needed for inhibition of its spores
- symptoms
- double vision, blurred vision, drooping eyelids, slurred speech, difficulty swallowing, dry mouth, and muscle weakness.
- If the disease is not treated, symptoms may progress to paralysis of the arms, legs, trunk, and respiratory muscles.
- Patients with severe cases that involve paralysis of the respiratory muscles may need mechanical ventilation and intensive care for weeks or months.
- Treatment
- Anti-toxin for infant botulism maintained by California Dept of Public Health.
- CDC maintains equine antitoxin for other botulism types
- Reduces severity if administered early
- Pentavalent (ABCDE) botulinum toxoid
- detection
- identification of toxins in the food involved
- injection of extracts of the food into passively immunized mice (mouse neutralization test).
- Takes 48 hours
- followed by culturing all suspect food in an enrichment medium, for detection and isolation of the causative organism.
- Detection of Type A, B, E, and F Clostridium botulinum Toxins Using Digoxigenin-labeled
IgGs and the ELISA (DIG-ELISA). - Digoxigenin-labeled antitoxin IgG’s
- anti-digoxigenin horse radish peroxidase conjugate (HRP)
- (TMB) is used for the HRP enzyme
- A, B, E, and F botulinal toxins are detected at approximately 10 MLD/mL (0.12-0.25 ng/mL)
10
Q
Domoic acid
A
- Domoic acid (DA) is a kainic acid-type neurotoxin that causes amnesic shellfish poisoning (ASP). It is produced by algae and accumulates in shellfish, sardines, and anchovies. When sea lions, otters, cetaceans, humans, and other predators eat contaminated animals, poisoning may result.
11
Q
Shigella species
A
- Closely related to E.coli based on DNA homology (same species?)
- 4 Species:
- S. sonnei (Subgroup D) most common in U.S.
- S. flexneri (Subgroup B)
- S. dysenteriae (Subgroup A) rare in U. S.
- S. boydii (Subgroup C) rare in U. S.
12
Q
Shiga toxin
A
- Shiga toxin is an exotoxin produced most commonly by Shigella dysenteriae type 1 (Most virulent) and shiga toxin‐producing E.coli (STEC)
- Shiga toxin has enterotoxic, cytotoxic, and neurotoxic effects, can damage the mucosa, villi and nerve cells
- Shiga toxin is heat‐stable, not inactivated by pasteurization, can be inactivated by autoclave and bleach
13
Q
Shigella spp. Characteristics
A
- 25g into 225mL of mBWP(modified peptone water media) + novobiocin
- General Characteristics
- Family Enterobacteriaceae
- Gram‐negative rods
- Non‐motile
- Non‐spore forming
- Facultative anaerobe
- shigellosis
- Shigellosis is an infectious disease caused by Shigella.
- Onset time: Usually 24‐36 hours (range from 1‐7 days)
- Symptoms: bloody diarrhea, fever, stomach cramps, nausea/vomiting
- Resolve in 5 – 7 days
- Epidemiology and public health significance
- Humans are the most common reservoir (infections have been observed in primates as well)
- Fecal‐oral transmission
- 10‐200 cells are needed for infection in healthy individuals
- Every year, Shigella is estimated to cause about 500,000 illnesses in the United States
- about 14,000 cases of shigellosis are reported to CDC
- Transmission
- Waterborne
- Foodborne
- Restricted to higher primates including humans
- usually spread among humans by food handlers with poor personal hygiene
- Foods most often incriminated: potato salad, shellfish, raw vegetables, and Mexican dishes
- Characteristics of Shigella
- Non‐motile
- Do not produce gas from glucose
- Non‐lactose fermenting (S. sonnei can ferment lactose)
- Non‐sucrose fermenting
- Do not hydrolyze urea
- Do not produce H2S
- Lysine decarboxylase negative
- Citrate negative
- Fragile organisms
- Isolation is very difficult because:
- Can be present in low numbers
- Very fragile, do not survive long outside of host (generally measured in days)
- Do not compete well with other organisms
- Selective Plating
-
Hektoen enteric (HE) Agar
- Lactose, sucrose and Salicin: Shigella do not ferment these carbon compounds, thus do not cause a color change on HE plates.
- Xylose lysine deoxycholate agar (XLD) Agar
- Shigella cannot ferment xylose, therefore pH remain alkaline in the medium, and the colonies remain red
-
MacConkey Agar (MAC) is a selective and differential medium designed to isolate and differentiate enterics based on their ability to ferment lactose.
- Selectivity: Bile salts and crystal violet inhibit the growth of Gram-positive organisms.
- Lactose allows for differentiation: Organisms that ferment lactose will produce acid and turn the colonies pink.
- Non‐fermenters will produce colorless or clear colonies.
- Typical Shigella colonies will appear translucent white or colorless on MacConkey Agar after 24 hours incubation
-
Hektoen enteric (HE) Agar
- Screening biochemical tests
- TSI= K/A‐
- Non‐lactose fermenter
- Non‐sucrose fermenter
- H2S negative
- Anaerogenic*
-
LIA= K/A‐
- Lysine negative
- Non-motile
- Shigella Confirmation
- Screening
- TSI, LIA, Motility
- Confirmatory
- API, VITEK
- Serology
- Screening
14
Q
Vibrio
A
- Enrichment Media
- APW or PBS
-
TSI
- VC= A/A
- V mimicus = K/A
-
Polymyxin B
- El tor = res
- classical = susceptible
- Characteristics
- Halophilic ‐ requires salt (Na+) for growth (most media have enough salt for V. cholerae and V. mimicus) 0.029 to 4.1%
- Motile by sheathed monotrichous or multitrichous polar flagella
- Oxidase positive (except for V. metschnikovii)
- Catalase positive
- Ferment D‐glucose but rarely produce gas
- Reduce nitrate to nitrite
- Grow on TCBS medium
- Gram-negative straight, curved, or comma-shaped rods
- Mesophilic (20‐45C)
- Facultative anaerobes
- EPI
- The following Vibrio species cause majority of all foodborne illnesses:
- V. parahaemolyticus
- V. cholerae
- V. vulnificus
- Vibrio species account for a significant proportion of human infections from the consumption of raw or undercooked shellfish
- Increased foreign travel
- Use of recreational water facilities
- The following Vibrio species cause majority of all foodborne illnesses:
- Cholera Disease
- Onset time: 1‐5 days; average 2 days
- Symptoms
- 75% asymptomatic
- Profuse watery diarrhea – “rice water stool”
- Vomiting
- Abdominal pain
- Dehydration
- Thirst
- Circulatory collapse
- Sunken eyes
- Death
- Virulence Factors
- Toxins
- CTX – Cholera toxin
- HAP protease – Haemagglutinin protease – extracellular enzyme
- RTX toxin – repeat‐in‐toxin
- ACE and Zot – accessory cholera enterotoxin and zonula occludens toxin
- Colonization factors
- TCP – Toxin co‐regulated pilus
- VPI – Vibrio pathogenicity island
- MSHA – Mannose‐sensitive hemagglutinin
- Toxins
- Incriminating Foods
- Polluted waters
- Foods washed or prepared with contaminated water
- Rice
- Raw vegetables
- Seafood
- Raw or undercooked shellfish
- Shrimp and crab
- Host factors
- Achlorhydria ‐ low gastric acid levels
- Children
- Elderly
- Use of antacids
- Blood Group O
- Most common presentation is gastroenteritis
- Vibrio parahaemolyticus
- Implicated Foods
- Raw shellfish and fish
- Salt-containing foods (i.e. salted vegetables)
- Cross-contaminated boiled shrimp or crab
- Treatment
- Not necessary in most cases
- In severe or prolonged illnesses, antibiotics such as tetracycline or ciprofloxacin can be used
- Vibrio vulnificus
- Onset time:
- Mean 16h: < 24 h usually
- Symptoms:
- Primary sepsis – Fever (~104F), chills, diarrhea, nausea, skin lesions, loss of consciousness, seizure, and death (can occur 2‐3 days following ingestions)
- Gastroenteritis (rarely occurs in a competent host)
- Cellobiose-Polymyxin B-Colistin (mCPC) Agar
- Thiosulfate‐Citrate‐Bile Salts‐Sucrose (TCBS) Agar
15
Q
E. coli
A
- General Characteristics
- Gram-negative, rod-shaped
- Motile
- Facultative anaerobe
- Model prokaryotic organism - host organism for the majority of work with recombinant DNA.
- Can be differentiated serologically based on 3 major surface antigens
- 173 O antigens, 80 K antigens and 56 H antigens
- The number of serotypes can be between 50,000-100,000
- STEC (EHEC) in food
- O157:H7
- Isolation from food
- Identification/confirmation methods
- Subtyping methods
- Groups
- Enterotoxigenic E. coli (ETEC);
- Enteropathogenic E. coli (EPEC);
- Enteroinvasive E. coli (EIEC);
- Enterohemorrhagic E. coli (EHEC);
- Enteroaggregative E. Coli (EAEC);
- Diffusively adherent E. coli (DAEC).
-
Enteropathogenic E. coli (EPEC)
- First pathotype of E. coli to be described
- Presence of locus of enterocyte effacement (LEE) pathogenicity island
- virulence factors- eae gene (intimin), tir gene (intimin receptor), and Type III Secretion System
- Allow adherence to intestinal epithelial cells by forming attaching and effacing lesions (A/E)
- Common cause of diarrhea in infants
- Mortality 25-50%
- Onset is rapid, as soon as 4 hours
-
Enteroinvasive E. coli (EIEC)
- Biochemically, genetically, and pathogenically related to Shigella spp.
- But unlike Shigella spp., does not produce any toxins
- Able to invade colonic epithelial cells which can cause mild diarrhea
- Adhesin proteins for binding and invading host cells
- Once inside able to invade adjacent epithelial cells
- Illness
- Mild form of bloody diarrhea similar to Shigella
- Self-limiting with no known complications
- Infectious dose 200- 5000 cells (higher than Shigella)
- Cellular invasion via endocytic vacuoles
- Biochemically, genetically, and pathogenically related to Shigella spp.
-
Enterotoxigenic E. coli (ETEC)
- Best known as the causative agent of traveler’s diarrhea
- Virulence factors that elicit a diarrheal response
- heat-labile (LT) toxin and heat stable (ST) toxin
- CFA/I and CFA/II (Fimbrial colonization factors)
- Major cause of diarrhea in infants in less developed countries
- 380,000 deaths worldwide attributed to ETEC every year (mostly among children)- WHO
- High doses ranging from 10 million to 10 billion ETEC cells needed in adults
- Illness
- Can cause profuse watery diarrhea, low-grade fever, and vomiting
- Usually mild and self-limiting, but severe forms last longer and resemble cholera
- Can last 21 – 120 days
- Causes nutrient malabsorption
- Enterohemorrhagic E. coli (EHEC)
- Important enteric bacterial pathogens in the United States (US)
- Serotype O157:H7 is the predominant strain
- Accounts for ~75% of all EHEC infections worldwide
-
Produce one or two Shiga toxins (stx)
- Stx1 nearly identical to toxin produced by Shigella dysenteriae Type 1
- Stx2 associated with HUS and kidney failure
- Presence of LEE pathogenicity island and enterohemolysin
- Some patients with EHEC infection develop hemolytic uremic syndrome (HUS)
- A severe complication characterized by renal failure, hemolytic anemia, and thrombotic thrombocytopenic purpura (TTP) that can be fatal (3 to 5% mortality)
- Most cases of Hemolytic Uremic Syndrome (HUS) in the US are caused by E. coli O157.
- Primarily affects children and the elderly
- Modes of Transmission
- Through contact with animals and their environment
- Farms, markets
- Through contaminated food and water
- Undercooked ground beef
- Spinach, Alfalfa sprouts, radish sprouts
- Apple Cider
- Salami
- Municipal and lake water
- person to person
- Through contact with animals and their environment
- E. coli O157:H7 Food Poisoning
- Onset time: 2-3 days or as long as 9-10 days
- Symptoms: severe cramps and diarrhea, bloody stool on the 2nd or 3rd day, possible vomiting and fever
- In most cases, infection resolves in 6-8 days
- Some patients develop HUS or TTP
- 1/3 of infected persons require hospitalization
- Isolation from food
- Enrichment
- Immunomagnetic Separation
- Selective plating media
- Identification/Confirmation: real-time PCR, Biochemical/Serologic tests
- Subtyping methods
- Enrichment Broths
- Modified Buffered Peptone Water with Acriflavin, Cefsulodin and Vancomycin
- E.coli enrichment Broth (EEB) with Cefixime, Cefsulodin and Vancomycin
- Modified EC Broth - Novobiocin
- Modified Trypticase Soy Broth - Novobiocin
- Selective Plating
-
MACCONKEY AGAR WITH SORBITOL, CEFIXIME, AND TELLURITE (CT-SMAC)
- E. coli O157:H7) form smooth and colorless colonies.
-
EMB = Eosin methylene blue agar
- the color is green metallic sheen. The green metallic sheen indicates E. coli is able to ferment lactose to produce strong acid endproducts.
-
MACCONKEY AGAR WITH SORBITOL, CEFIXIME, AND TELLURITE (CT-SMAC)
- ID
- Real-time PCR
- uidA gene (encodes for -glucuronidase, probe specific for O157:H7)
- Stx1 and Stx2
- Conventional Biochemical tests
- MUG disk test = +
- Indole = +
- Oxidase = -
- Motility = +
- Vitek 2 and API *
- Serologic Tests
- somatic and flagellar antigens
- E. coli O157:H7 is motility (+)
- E. coli O157:H7 is indole (+)
- E.coli O157:H7 is oxidase(-)
- Real-time PCR
16
Q
Yersinia
A
- Pyrazinaminadase = Negative
- Esculin = Negative except biotype 1A
- TSI
- enterolytica = A/A - -
- pseudotb = K/A-
- LIA
- enterolytica = k/a - -
- salmonella = K/K + -
- Urea
- Enterolytica = urease Positive
- E. coli = -
- Enrichment
- PSBB
- FOODS
- Strains of Y. enterocolitica can be found in meats (pork, beef, lamb, etc.), oysters, fish, crabs, and raw milk. However, the prevalence of this organism in soil, water, and animals, such as beavers, pigs, and squirrels, offers many opportunities for Yersinia to enter the food supply.
- Y. enterocolitica/pseudotuberculosis = Oral ingestion
- Human Pathogens
- Y. pestis = rodents/fleas
- Y. enterocolitica = pigs/rodents
- Y. pseudotuberculosis = lagomorphs/birds
- Yersinia enterocolitica = YERSINIOSIS
- Gastrointestinal syndromes
- Enteritis
- Enterocolitis
- Ileitis
- Mesenteric lymphadenitis
- Septicemia
- Miscellaneous infections
- Reactive arthritis
- Pharyngitis
- Endocarditis
- Gastrointestinal syndromes
- Yersinia – General Properties
- Straight rods to coccobacilli
- Non-motile at 37°C. Mostly motile at < 30°C
- Facultative anaerobes (at 4 to 43°C)
- Most carbohydrates fermented with little or no gas production
- Y. enterocolitica - Isolation
- Isolation can be problematic from stools:
- XLD, and HE are unsatisfactory
- Produces small or pinpoint colonies on typical enteric media
- can be overgrown by rapid-growing species
- selective medium should be included when yersiniosis is suspected
- Cold enrichment is not recommended
- recover non-pathogenic Yersinia or pathogenic strains present only as colonizers
- Transport:
- Cary-Blair, Amies, Stuart’s, or buffered glycerol in saline
- Isolation can be problematic from stools:
-
Cefsulodin Irgasan Novobiocin Agar (CIN)
- CIN selective reagents:
- Cefsulodin/irgasan inhibit GN bacteria
- Novobiocin inhibits GP bacteria
- D-mannitol provides differential characterization
- Incubate at 25°C; look for “bulls-eye” colonies
- Few non-Yersinia genera grow on this media but larger
- Citrobacter
- Serratia, Morganella
- Pick isolates to TSI, LIA, MIO
- CIN selective reagents:
-
Under Title 17, CCR, Section 2505:
- List (e) (2) “Yersiniosis (Yersinia spp., non‐pestis) (isolation from a clinical specimen)
- Report to local or State PHL within one working day
17
Q
Salmonella
A
- Isolation and Identification of Salmonella in food
- The FDA Bacteriological Analytical Manual (FDA-BAM)
- The USDA Microbiology Laboratory Guidebook
- A real-time PCR method developed in 2008
-
Selective Plating on Hektoen Enteric (HE) Agar
- Selective inhibition
- Bile salts inhibit gram-positive organisms and reduce some gram-negative organisms other than Salmonella and Shigella.
- Differential ability
- Non-lactose fermenting Salmonella produces blue to green colonies with black precipitates due to H2S production.
- Selective inhibition
-
Xylose Lysine Deoxycholate (XLD) Agar
- Selective inhibition
- Sodium deoxycholate inhibits the growth of gram-positive organisms.
- Differential ability
- Xylose-fermenting and lysine decarboxylase-positive Salmonella produces red colonies with black precipitates due to H2S production
- Selective inhibition
- ID
- TSI = Glucose(+);Lactose/Sucrose(-); H2S(+) Salmonella = K/AG+
- Lysine Iron Agar (LIA) = Deamination(-);Decarboxylation(+); H2S(+) Salmonella
- Urease(-) = Salmonella
- Motility = Pos
-
3 major antigens
- O (somatic) antigen
- H (flagellar) antigen
- Vi (capsular) antigen
18
Q
Listeria
A
- incubation 1- 4 weeks
- systemic flu like symptoms endo carditis
- neonatal
- L. Mono 10 to 100million CFU
- days to week duration
- TSI
- A/A
- General
- Motile with peritrichous flagella at <30C
- Not motile at 37°C
- Psychrotropic, 0 to 45°C
- pH range 4.4 to 9.6
- Halotolerant‐ 10% ‐12% NaCl
- Destroyed by heat, 60°C
- Able to form biofilms
- Virulence factors
- Listeriolysin A
- Phospholipase
- Surface proteins (i.e. Inl, ActA
- 15 serotypes that can cause disease
- 3 serotypes account for >95% of isolates
- 1/2a, 1/2b‐ Sporadic infection
- 4b‐ Mainly responsible for epidemic foodborne outbreaks in Europe and North America since the 1980s
-
Sources
- Susceptible Foods
-
RTE= Ready to eat
- Hot dogs
- Deli meat
- Soft cheeses
- Raw food
- Meat
- Fruits
- Vegetables
- Unpasteurized foods (i.e. dairy)
- Smoked seafood
- In the U.S., Listeria infections cause
- 1600 illnesses annually, with 260 deaths
- 90% cases result in hospitalization
- 20‐30% mortality
- 3rd leading cause of foodborne death in the U.S.
- ID
- Buffered Listeria Enrichment Broth
- Supplements
-
Nalidixic acid: inhibits Gram-negative spp.
- Does not ininhibit Pseudomonas or Proteus spp.
- Acriflavine: inhibits many other Gram (+) bacteria, parasites, and fungi
-
Cycloheximide: inhibits yeasts and molds
- Very toxic
- Can be substituted by pimaricin (a.k.a. matamycin)
- CHROMagar Listeria
- RAPID’L. mono = blue halo colonies
- Motility = Umbrella type at room temp
- Catalase = Positive
- oxidase negative,
- fermentation of carbohydrates to acid but not to gas,
- hydrolysis of esculin and sodium hippurate,
- methyl red positive,
- ammonia production from arginine,
- negative reaction for hydrogen sulfide production,
- indole negative,
- nitrate reductase negative,
- no gelatin liquefaction,
- no hydrolysis of starch and
- no urea hydrolysis.
- Beta‐lysin disk test‐ CAMP test
- Enhanced hemolytic activity through synergistic interactions with CAMP factor
-
PCR
- Streak presumptive colonies from chromogenic plates onto TSA‐YE agar plates to obtain pure isolates
- 2° PCR confirmation
- Subtyping through - Whole genome sequencing of pure isolates‐ Illumina technology
- Listeria monocytogenes is an important intracellular pathogen, causing illness in humans and animals
- Transmission is primarily through consumption of contaminated raw or RTE foods
- Listeria infections are rare, but serious, predominantly affecting susceptible populations
- Virulence factors are key in establishing infection and pathogenicity
- Molecular and biochemical methods are used to detect and isolate Listeria
20
Q
Campylobacter
A
- Campylobacter Enrichment Broth (Bolton formula) in microaerophilic conditions @ 42 and 35 degrees
- H2S @ 35 but not 42 for jejuni
- Nitrate POS
- Hippurate Pos
- Catalase Pos
- Oxidase Pos
- Member of the family Campylobacteraceae which also includes:
- The genus Arcobacter
- The genus Helicobacter
- Currently, there are approx. 30 species in the genus; some originally belonging to the genus have been transferred to either Helicobacter or Arcobacter
- The most important species in the genus currently include:
- C. jejuni-•leading cause of bacterial gastroenteritis in the U. S. (thermophilic)
- C. coli
- C. fetus-•primarily associated with bacteremia and extraintestinal infections in patients with underlying diseases. Does not grow at 42C.
- C. upsaliensis
- C. concisus
- Most common clinical presentation: acute gastroenteritis
- Chief symptoms: acute watery diarrhea, often with fever, occasionally with dysenteric characteristics (blood)
- Incubation period: 2 – 5 day
- Infectious dose: 500 – 1000 cfu
- Illness usually self-limiting
- Relapse occurs in 5-10% of patients
- Treatment
- 4 Fluid and electrolyte replacement
- 4 Azithromycin is the drug of choice
- 4 In 5-10% of patients, relapses occur, if untreated
- GUILLAIN-BARRE SYNDROME - Most common bacterial cause of GBS; 30% preceding paralysis
- REACTIVE ARTHRITIS-2 -5 % pts post-Camp infection
- IRRITABLE BOWEL SYNDROME- 33% pts post-Camp infection
- Most common cause of gastroenteritis in the US
- Campylobacter - Microbiology
- Gram-negative bacilli
- Curved, S-shaped, or spiral rods
- Motile by polar flagella
- Microaerophilic (5% O2, 10% CO2, 85% N2)
- Produce gray, flat, irregular and spreading colonies
- Difference in appearance due to horse blood, hydrogen and moisture content.
- Direct plating of specimen to selective media
- Microaerophilic environment; culture in gas generator packs such as Gas-pak
- Cultured at thermophilic temperatures (42°C) for recovery of C.jejuni/C.coli
- No culture conditions recover all Campylobacter species satisfactorily
- To optimize recovery plate to two selective media, one being blood-free. Increases yield up to 15%.
- Blood-free media such as Charcoal Cefoperazone Deoxycholate Agar (CCDA), Charcoal-based Selective Medium (CSM), or Karmali agar = Spread out grey colonies
- CEFEX laked blood
- Blood-containing media such as Skirrow’s medium (lysed horse blood) and Campy-CVA (cefoperazone, vancomycin, amphotericin)
- Sheep blood (10%)-based agar + antibiotics such as Butzler’s or Blaser Media
- Principle: Campylobacters (thin and highly motile) pass through membrane filters easily (0.45 – 0.65 μm), whereas larger stool flora are retained
- Hippurate Hydrolysis
- To distinguish between C. jejuni and C. coli.
- Gas Liquid Chromatography (sensitive & specific)
- Hippurate broth + formic acid + fumurate acid
- Incubate for 5 days (42oC)
- Observe for benzoic acid peak
- Chemical Test – detect production of glycine endproduct (2hr)
- Use of nonspecific ninhydrin indicator to detect the presence of an amino acid.
- 48 hr test detects benzoic acid precipitated by ferric chloride
- Campylobacter Identification
- Conventional
- Growth at 42°C on selective media
- Gram stain appearance;
- positive oxidase reaction
- Rapid hippurate hydrolysis (C. jejuni)
- Hippurate-negative, indoxyl-acetate-positive (C. jejuni/coli)
- CIDT
- Immunoassays (ProSpecT, Premier Campy, Immunocard STAT! CAMPY) detects surface antigen common to jejuni – coli
- PCRs (BioFire FilmArray GI, xTAG, ProGastro SSCS)
- Molecular
- 16S rRNA gene sequencing (1500 bp)
- MALDI-TOF
- •Most laboratories do not report beyond genus identification
- Some report as C. jejuni, C. jejuni/coli, or Campylobacter species
- Conventional biochemical tests unsatisfactory in species identification beyond these groups
- Hippurate positive = C. jejuni subsp. jejuni
•Hippurate negative = C. jejuni/coli
•Hippurate positive, nitrate negative, Ceph S = C. jejuni subsp. doylei
- Prevention
- Strict control of access to food producer establishments by unauthorized persons, includings birds, rodents and other animals
- insect control
- worker’s control [hygiene barriers]
- Water purifying [chlorination
