Systemic Bacteriology Food Borne Infections-Handout

Module- 2
Systemic Bacteriology &

Food born diseases
By: Mengistu A. (MSc, Asst Professor in Medical Microbiology)

Objective
• At the end of this chapter, students will be able to:

• Discuss what is food-borne disease
• Differentiate food intoxication and food infection.
• Describe MOs responsible for food borne illnesses.
• Identify the original sources of MOs and the type of food stuffs
• Discuss pathogenicity, clinical disease manifestations

and epidemiology
• Discuss diagnosis, treatments, prevention and control
of the bacteria
2
Food born illnesses
❑Food-borne illness is also called food poisoning caused by

eating contaminated food.
❑Annually, an estimated of10% of the population were affected by
food-borne illness, which leads for annual cost of treatment and
work hours lost at more than $30 billion.
❑“Everybody will get a food-borne illness twice per year”
❑When we discuss food poisoning, we must distinguish between
food intoxication and food infection.
• Food intoxication
• Food infection ???? Definition
3
Food born illnesses……
❑Infectious organisms or their toxins can contaminate food at

any point of production: growing, harvesting, processing,
storing, shipping or preparing.
❑Cross-contamination — the transfer of harmful organisms

from one surface to another — is often the cause.
4
Common Foodborne Pathogens
▪ Infectious organisms — including bacteria, viruses and
parasites — or their toxins are the most common causes of
food-borne infections.
▪ Among microbes mainly bacteria are involved in food-borne
illness
Bacteria Viruses Parasites
o E. coli 0157:H7 o Norovirus o Cryptosporidium
o Salmonella spp. o Rotovirus parvum
o Staphyloccus aureus o Hepatitis A
o Listeria monocytogenes o Giardia lamblia
o Campylobacter Spp. o Cyclospora
o Shigella spp.
o Clostridium spp.
5
Illness Mechanisms
• Infection
• Microorganisms are ingested and then cause illness
• Intoxication
• Toxins are produced by the pathogen, usually in the food. When food is
consumed, illness occurs.
• Even if microorganisms are killed, toxin can still remain in the food
6
Bacteria Involved in Food born
illnesses and other infections
7
I. Staphylococci
General characteristics
Gram positive, non- spore forming, non- motile, spherical shaped and arranged
in grapelike clusters
Grow in ordinary media (aerobic or facultative)
Staphylococci make a very large contribution to man's

commensal flora and also account for a high proportion of
his acute and chronic suppurative lesions.
Food poisoning: Heat stable stap.cocci enterotoxin
8
Staphylococci …
❑The genus has at least 30 species

❑ The three main species of clinical importance are:
▪Staphylococcus aureus
▪Staphylococcus epidermidis
▪Staphylococcus saprophyticus
 Some of them are normal flora of skin and mucus membrane like nasal cavity,
vaginal mucosa etc.)
 The pathogenic staphylococci often hemolyze blood, coagulate plasma, and
produce a variety of extracellular enzymes and toxins
9
Staphylococcus aureus
• Causes food intoxication.
• This bacterium produces 6 serologically different
enterotoxins (A, B, C, D, E & F), which are heat resistant.
• Found most commonly in cheese, Meats, milk, cream,
various salads, and in foods prepared by hand,
• Symptoms usually appear in 2-5 hours after a meal, but
they may appear sooner.
• The patient will experience nausea, vomiting, abdominal
cramps, and diarrhea.
10
Pathogenesis
❑ S. aureus is a major pathogen for humans

❑ Almost every person will have some type of S. aureus
infection in a lifetime, ranging from food poisoning or
minor skin infections to severe life-threatening infections
❑ Pathogenesis of Staphylococci is combined effect of:

• Extracellular factors (structural components)
• Toxins productions
• Invasive properties of the strain
11
Antigenic structure Staphylococci
1. Structural components of cell wall :
• Antigenic polysaccharides on cell wall of Stap.cocci:
• Peptidoglycan: Pathogenesis of infection & endotoxin like
activity
• Teichoic acids: used as attachment organ and can initiate host
immune response
• Protein A: inhibits antibody-mediated clearance by binding IgG

• Other cell wall components, like capsules, slime layer,,,,,
N.B: * Capsules inhibit Phagocytosis by leukocytes
12
Antigenic structure ….
2. Enzymes productions
A. Catalase
• Converts H202 into H20 & 02
• Staphylococci --- Catalase positive
• Streptococci ---- Catalase negative
B. Coagulase
• Produced by S. aureus only
• Clots oxalated/citrated plasma by binds to fibrinogen &
• Deposit or aggregate fibrin on the surface of stap.cocci
• Inhibits phagocytosis
C. Other enzymes
• Hyaluronidase: Hydrolyzes hyaluronic acids in connective tissue,
promoting the spread of staphylococci in tissue
• Staphylokinase: Cause fibrinolysis
• Beta- lactamase: Disrupts the Beta -lactam rings of antibiotic
13
3.Toxins productions
A. Exotoxins (cytotoxins (α, β, γ))
▪ Toxic for many cells, Lyse RBCs, damage platelets &vascular

smooth muscle and cause necrosis in skin
B. Enterotoxins (6 (A-F):
▪ Resistant to heat and gut enzymes, soluble toxins produced by S.
aureus
▪ Bind to MHC class II molecules and inhibit T- cell activation
▪ Responsible for staphylococcal food poisoning when grows on
carbohydrate & protienous food and increasing intestinal peristalsis
and fluid loss; cause diarrhea & vomiting
14
C. Leukocidin: This toxin of S. aureus kills WBCs
D. Exfoliative toxin (ETA, ETB)
▪ Serine proteases that cause staphylococcal scalded skin
syndrome; generalized desquamation of epidermis in young
children
E. Toxic shock syndrome toxin
▪ Produced by mostly S. aureus
▪ Super antigen (stimulates proliferation of T cells and release
of cytokines); produces leakage or cellular destruction of
endothelial cells--Associated with: fever, shock and skin
rash
15
Epidemiology
❑S. aureus is normal flora on human skin & nose (25 – 75% carriers)
❑They are hardy, being resistant to heat and drying, and thus can
persist for long period on inanimate objects
Transmission
– Direct contact or exposure to contaminated fomites/
inanimate objects (bed, clothing, linens)
Risk factors includes;
–Use of foreign bodies (sutures & intravenous catheters)
–Previous surgical procedure
–Use of antibiotics that suppress normal flora
16
Clinical significance of S. aureus
❑Inflammatory disease (Superficial Skin infection):- mainly

pyogenic (pus forming) infections such as impetigo, follicullitis,
furuncles (boils), carbuncles, surgical wound infections, burn
infections
❑Other diseases (deep-seated lesions):- osteomyelitis
(important infection), bronchopneumonia, conjunctivitis,
septicemia, endocarditis, septic arthritis
❑Toxin mediated disease
– Food poisoning
– Toxic shock syndrome
– Scalded skin syndrome
17
Staphylococcus aureus….cont’d…
❑The source of this bacterium is human carriers, who can

represent 25% or more of the population, will have this
bacterium in the nasopharynx.
❑The illness is self-limiting within a day or two and not fatal,

unless complications arise.
❑Severity of the symptoms depends on the amount of toxin

ingested and the individual’s resistance.
18
Staph skin infections
Superficial folliculitis Deep folliculitis Furuncle (infected hair follicle
Carbuncle: Multiple abscesses Staph impetigo Scalded skin syndrome

around many hair follicles
19
Methicillin-resistant Staphylococcus aureus (MRSA)
❑Healthcare-associated MRSA: Highly resistant to

commonly used antibiotics such as Methicillin (= all β-lactam
antibiotics), Tetracycline, Co-trimoxazol, rifampin, clindamycin, macrolides,
quinolones
❑Cause nosocomial infection such as surgical wound infections,
urinary tract infections, bloodstream infections, and pneumonia
❑Risk factors included:
– Prolonged hospitalization
– Prolonged antimicrobial use
– Stay in an intensive care or burn unit
– Exposure to a colonized/infected person
– Residence in a nursing home
– Age >65
– Chronic dialysis
20
Community-acquired MRSA:
❑New type of MRSA identified– Infection in a person with no

prior history of health care exposure, i.e. hospitalization,
surgery, permanent devices or hemodialysis
❑It is resistant to certain antibiotics, including methicillin,
oxacillin, penicillin and amoxicillin.
❑Risk Factors for CA MRSA Infection
▪ Child under the age of 2
▪ Male with history of having sex with men
▪ Shaving of body hair, especially extensive shaving of arms
and/or legs related to sports participation
21
How is MRSA transmitted?
▪ Person to person via hands & skin-to-skin contact

▪ Sharing contaminated items such as soap, towels, clothing,
athletic equipment, razors and other personal care items
▪ Contaminated surfaces
▪ Breaks in skin, abrasions increase risk of transmission
❑Factors that make it easy for MRSA to be transmitted (5 C’s)
▪ Crowding
▪ Frequent, skin-to-skin Contact
▪ Compromised skin (abrasions, cuts)
▪ Contaminated surfaces
▪ Lack of Cleanliness
22
Laboratory diagnosis of Staphylococci
❑Specimens: skin swab, pus, sputum or tracheal aspirate, blood

for culture, CSF, stool, vomits and remains of food
❑ Microscopy (Stained smears): gram positive cocci in clusters
seen. Not helps to distinguish among different Spp.
Gram staining preparation of pus: Gram positive cocci in grapelike

clusters.
23
Growth characteristics……..
❑On Blood Agar : Staphylococcus spps form slightly raised

and easily emulsified, white to creamy colonies, with most
strain show β- hemolysis colonies.
❑Mannitol salt Agar (8-10% NaCl): S. aureus grow well and

forms golden yellow colonies
➢ S. epidermidis and S. saprophyticus form gray to
white colonies
24
Biochemical reaction
• Catalase Test: breakdown of H2O2 Catalase

2H2O2 2H2O + O2
to H2O & O2 giving bubbles (Gas bubbles)
formations if the bacteria is Staphylococci - Positive
catalase enzyme producers. Streptococci – Negative
▪The catalase test can also be done on a slide. Remove a

colony, place it in a drop of H2O2 & watch for bubbles.
25
Biochemical reaction……
▪Coagulase test: causes plasma to clot by

converting fibrinogen to fibrin clot.
▪ Slid test: emulsify few similar colonies of in a drop of
saline and stir with straight wire that dipped into
plasma. Observe for clumping within 10 seconds. If the
slide test is negative, you should do a tube test.
▪ Tube method:
▪ Human / citrated rabbit plasma diluted 1:5 (+) equal
volume of growth colonies from broth culture
• Incubated at 370C
• Positive test: If clots form in 4hrs S.
aureus
26
Biochemical reaction…
❑Sensitivity testing:
• Disk diffusion susceptibility testing (Mueller Hinton agar
with Novobiocin)
• Novobiocin sensitive… S. aureus and S. epidermidis
• Novobiocin resistant……S. saprophyticus
❑S. aureus is also distinguished from most coagulase-
negative staphylococci by being mannitol-positive.
❑Serological Testing
• Anti-bodies to teichoic acid (Chronic infections)
27
Prevention and control
▪ Proper disinfection and sterilization of medical devices
▪ Contact precautions should be used for all patients with

known MRSA infections and for all patients with skin/soft
tissue infections
▪ Use of proper personal hygiene
▪ Early treatments of infected persons
▪ Early detection and screening of high risk groups
▪ Increased awareness by healthcare providers
28
I. The Genus Bacillus
General Characteristics
❑ It is large, spore-forming, aerobic and facultative anaerobic gram-positive
rods
❑ Endospores is oval and centrally located
❑ Some are non- motile and has capsule that is anti- phagocytic
❑ They are thermophilic, psychrophilic, acidophilic, alkalophilic, halotolerant,

halophilic
29
The Genus Bacillus….
❑ Majority are harmless and saprophytic and widely

distributed in natural environment (mainly in soil and
water)
❑ Some opportunistic or obligate pathogens of animals
❑ At least 48 species are known but only B. anthracis and
B. cereus cause disease in humans.
❑ B. cereus can produce enterotoxin and cause food
poisoning
30
Bacillus anthracis…….
❑The pathogenicity of B. anthracis depends on two

virulence factors: polypeptide capsule and production of
toxin (edema & lethal toxin)
❑Toxins produced consists of three proteins: Protective

antigen (PA), lethal factor (LF) & edema factor (EF)
▪ EF with PA forms edema toxin
▪ LF plus PA form lethal toxin, which is a major virulence factor
and cause of death in infected animals and humans
31
Clinical significance
❑B. anthracis is the principal pathogen of the genus which

causes anthrax — primarily a disease of herbivores– goats,
sheep, cattle, horses, etc
❑In humans, the infection acquired by entry of spores

through;
– Injured skin (cutaneous anthrax)
– Rarely the mucous membranes (gastrointestinal anthrax)
– By inhalation of spores into the lung (Pulmonary anthrax)
32
Clinical significance……
1. Cutaneous Anthrax (‘malignant pustules’)
– It is the commonest form of anthrax
– Bacilli enter damaged skin, producing a
blister (‘malignant pustule’) which
usually results in painless ulcerates with
black (necrotic) center and edematous
margin
– About 20% mortality rate in untreated
cases
33
Clinical significance…….
2. Pulmonary anthrax (wool sorter’s disease)
‒ Caused by inhalation of spore
– Initial symptoms resemble common cold
– Progress to severe breathing problems and shock
– characterized by hemorrhagic pneumonia and
lymphadenitis.
– Usually results in death 1-2 days after onset of acute
symptoms
– Treatment usually not effective after symptoms are present
– Mortality rate 99% in untreated cases
34
Clinical significance…….
3. Gastrointestinal (Enteric) Anthrax
◼Caused by ingestion of contaminated meat from infected
animals
◼Abdominal pain, fever, vomiting, bloody diarrhea
◼Septicaemia often develops with meningoencephalitis
complication
◼25% to 60% mortality rate
4. Bacteremic anthrax
◼Presents with clinical features of sepsis
◼Prevalent in Africa including Ethiopia
◼It is the threat of biological warfare
35
Bacillus cereus
❑This bacterium is :
• B. cereus is a pathogen that produces enterotoxin and cause

disease that is more an intoxication than a food-borne
infection.
• Gram positive, aerobic, and spore-forming rod.

• Found in most soils, thus it has easy access to food.
• Can be controlled by keeping food colder than 10C or

warmer than 55C.
36
Bacillus cereus……cont’d…
❑Causing two types of gastrointestinal illness, depending on

the type of food involved and upon ingestion of the
enterotoxin.
▪ The emetic (vomiting) type, associated with ingesting
food (fried rice) contaminated with pre-formed toxin
▪ The diarrheal type, associated with ingesting large
numbers of bacterial cells and/or spores in
contaminated food -meat dishes and sauces
37
Bacillus cereus……cont’d…
❑This bacterium mostly found in: Meats, Vegetables, Sauces

and rice.
❑Symptoms include :
• Abdominal cramps, Profuse watery diarrhea and
sometimes nausea and vomiting.
❑Recently food infections involving this bacterium have been
reported.
❑Such outbreaks were associated with a high number of B.
cereus cells (>1x105/gm), implying the food must have been
temperature abused.
38
Bacillus cereus……
❑In addition, B cereus is an important cause of eye infections, severe
keratitis, endophthalmitis, and panophthalmitis.
❑Typically, the organisms are introduced into the eye by foreign

bodies associated with trauma.
❑B cereus has also been associated with localized infections and with
systemic infections, including endocarditis, meningitis, osteomyelitis,
and pneumonia
❑The presence of a medical device or intravenous drug use
predisposes to these infections.
39
Laboratory diagnosis
❑Specimens – pus or fluid or swabs from cutaneous lesions,

sputum, blood and spinal fluid based on site of infections
Microscopic
• Gram- stained smears from the local lesion or of blood
from dead animals often show chains of large gram-
positive rods with centrally located spores from culture
• Loeffler’s polychrome (McFadyean) methylene blue stained smear: Square
ended blue-black bacilli surrounded by a pink/purple
capsule.
40
Cultural tests
❑On blood agar: the B. anthracis produce non-hemolytic

gray to white mucoid colonies with irregular borders
❑ Mannitol egg-yolk phenol-red polymyxin agar (MYPA) is

recommended as a selective medium for the isolation of B.
cereus from faeces, vomit, or food. B. cereus gives a strong
lecithinase reaction.
❑ On Gelatin stab culture: It rapidly liquefies gelatin stabs.
41
Biochemical test:
❑Ferment glucose, maltose, sucrose, trehalose, and dextrine,

with acid production but not gas
❑Positive for Nitrate reduction test, Catalase test, starch

hydrolysis, VP, and gelatine liquefaction
❑ B. cereus, unlike B. anthracis, is motile, non-capsulate, and

produces haemolytic colonies on blood agar.
Serological test
• ELISA measure antibodies against edema and lethal toxins
42
❑Disposal of animal carcasses by deep burial or burning
❑Decontamination of animal product
❑Protective clothing and gloves for handling potentially

infected materials Eg. Animal’s hair, hide bone
❑Active immunization of domestic animals with live

attenuated vaccine
❑ Vaccination of workers in high risk occupation with anthrax

toxoid E.g. Leather factory
43
II. Genus Clostridium
❑ Most species of clostridia are anaerobic gram-positive

rods and spore forming (central, sub-terminal or terminal
spores)
❑ Majority are motile, but C. perfrengens is non-motile
❑ Most species are saprophytes found in soil, water, decaying

animal and plant matter but a few are pathogens to human
and cause tetanus, botulism, pseudo-membranous colitis,
food poisoning and gas gangrene
44
Genus Clostridium……
❑Some of them found in human and animal skin, intestinal
tract, and feces of animals
❑Most of them ferment organic compounds such as sugars with
acid, alcohols, gas production
• Foul smelling products from fermentation of aminoacids and fats
❑Produce extra-cellular enzymes which has role in invasion and

pathogenesis
❑ Three medically important species:
Clostridium botulinum
Clostridium perfringens
Clostridium tetani
45
I. Clostridium botulinum
• C. botulinum is :
• Gram positive, Terminal spore-forming rod.
• Obligate anaerobic,
• Motile and occur as single cells or in small chains
• Produce neurotoxin/Botulism toxin which is the
most potent toxin known
• Route of entry is under cooked consumption of
toxin contaminated spiced, vaccum packed or
canned food
46
Clostridium boutulinum…. Cont’d…
❑Based on the serological characteristics of the neurotoxin -

C. botulinum is divided into seven groups (A, B, C, D, E, F, &
G)
❑Type A,B,E are the concerns of food industry.
❑200 known outbreaks of botulism was observed in the

early 1900s, of which 10% was due to commercial products.
47
Clostridium boutulinum…. Cont’d…
❑Clostridium botulinum cause food intoxications (food
born botulism)
❑The toxin is absorbed from the gut and block
neurotransmission at peripheral synapses by preventing
the release of acetylcholine which results in flaccid
paralysis (blurred vision, swallow & Speech difficulty)
❑Death may occurs from respiratory paralysis or cardiac
arrest
❑It also cause infant botulism, wound botulism
48
Symptoms of Boutulinum
➢Appear in 12 to 24 hours after a meal. But, may vary

from a few hours to several days.
➢Initially, some vomiting & diarrhea, but no fever.
➢Blurred or double vision,
➢Difficulty in swallowing and speaking
➢Loss of muscle coordination.
➢Patient will be conscious to the very end
➢Death may occurs from respiratory paralysis or cardiac
arrest
49
II. Clostridium perfringens
❑C. perfringens is :
▪ Gram positive, anaerobic, non-motile, spore-forming
rod.
▪ Found in soil, thus is easily introduced into food.
▪ Growth range is between 16 and 50C
▪ A very short generation time (about 10 min) at higher
temperature limits.
50
Clostridium perfringens…. cont’d…
❑Produce Toxins: Phospholipase C (alpha- toxin or lecithinase)
– Can split lecithin w/c is components of Cm- leading to
hemolytic, cytolitic, necrotic effect on tissue.
– Cause soft tissue infections (A1) (Gas gangrene/ myonecrosis
& anaerobic cellulitis) and acute food poisoning (A2) with
secretory diarrhea due to release of enterotoxin in the
intestine
❑There are 5 types (A-E) on the basis of toxin production
❑C. perfringens type A-2 responsible for food infections or
intoxication.
51
Clostridium perfringens…. Cont’d…
❑Is responsible for food infection and may be intoxication

❑Meats, stews, sauces, meat pies, and etc are involved.
❑During cooking, oxygen is driven off and while vegetative
cells may be destroyed, the spores not only survive, but are
stimulated to germinate.
❑As the food passes though temperature ranges at which the
bacterium can grow, during refrigeration and later during
reheating, this bacterium can grow rapidly, reaching very
high numbers (1x106 or more/gm).
52
❑When ingested, the stomach acids may be neutralized by

the food, allowing the vegetative cells to survive.
❑As the food enters the small intestine, the cells begin to
multiply and starts to releasing an enterotoxin.
❑The incubation period is about 13 hours, but it can be

shorter or longer.
53
❑Symptoms include diarrhea, abdominal cramps as well as

headache and nausea.
❑Some patients may develop a fever, pass bloody stools,

vomit, or experience dizziness.
❑Younger patients have milder symptoms than older

patients.
54
❑C. botulinum: isolating the organism or detecting the toxin in
food product, patient faeces or serum
❑Culture: C. botulinum may be grown from food remains and
tested for toxin production, but this is rarely done and is of
questionable significance.
▪ C. botulinum is a strict anaerobe, grows best at 30–35 ºC.
❑Toxin can often be demonstrated in serum from the patient,
and leftover food by hemagglutination or radioimmunoassay.
55
C. botulinum……
❑Robertson’s cooked meat medium (RCMM):
▪ Types A, B and F blacken and digest cooked meat medium
(proteolytic reaction) and produce hydrogen sulphide
❑Blood agar subculture from RCMM (anaerobic culture):
▪ Most strains are beta-haemolytic, large semi-transparent
colonies with a wavy outline.
• Lactose egg yolk milk agar:
▪ C. botulinum hydrolyze lipid and protein in milk (lipase & proteinase +ve)
▪ Types A, B, and F show proteinase activity (area of clearing around the
colonies) due to the breakdown of casein in the milk by proteinase.
▪ Hydrolyze gelatin, ferment glucose but NLF
56
Laboratory diagnosis……C. perfringens
▪Specimen: Infected tissue, pus, vomits, left over food, serum

for Microscopic & culture examination
▪Grams stained smear
–Non-motile, capsulated, thick brick-shaped gram positive rods
in smears from tissue; spores are rarely seen.
Culture:
❑ Blood agar medium
➢ Produce β-hemolytic colonies in anaerobic atmosphere and
some strains double zone of hemolysis.
57
Culture:……..
❑Chopped meat-glucose medium and thioglycolate medium

–Sacharolytic bacteria reddening of the meat with rancid
or rotten egg small due to CHO fermentations with gas
Biochemical Tests:
▪ It ferments many carbohydrates with acid & gas
productions
– Lactose fermentation: Reddening of the medium
– Clostridia are catalase and oxidase negative.
58
Biochemical tests…
❑The most useful biochemical reactions which help to

identify C. perfringens and other pathogenic Clostridium
species can be tested by culturing the organism
anaerobically on lactose egg yolk medium
❑This medium tests for lecithinase C activity, lipase

hydrolysis, lactose fermentation and proteinase activity.
59
C. botulinum
– Maintain food in acid PH or storing at 4oC
– Heating at 80oC for 20 minutes (toxin is heat labile)
– Treatment (antitoxin, penicillin)
C. perfringens
—Proper wound care and use of prophylactic antibiotics
will prevent most infection.
—Adequate cooking of food
60
Clostridium tetani
❑Longer gram-positive rods with round terminal spores

giving characteristic “drum-stick”/tennis racquet
appearance.
❑Non- capsulated, most are motile with peritrichous flagella
❑Horse and humans are susceptible hosts, and cause tetanus
❑Tetanus is a non-invasive disease occurring because of the
release of exotoxins.
61
Virulence determinants and Pathogenesis
❑ Clostridium tetani produce two types of Tetanus toxin (exotoxins)
▪ Tetanolysin (hemolysin): Hemolytic property
▪ Tatanospasmin (neurotoxin): Plasmid encoded, heat & O2 labile
spasmogenic toxin causes paralysis by binding to motor nerve endings
(gangliosides); blocking the release of inhibitory neurotransmitter
(glycine); uncontrolled muscles contraction, muscle spasms and
convulsions (lockjaw) (spastic (rigid) paralysis)
▪ Cardiac failure can lead to death in approximately 55-65% of affected
persons.
62
Transmission
 Tetanus results from trauma or a puncture wound leading to tissue contamination.
63
Clinical significance of C. tetani
❑Generalized tetanus: affects masseter muscles

(trismus, lock jaw) & muscles of the back
❑Localized tetanus: involves muscles in area of
primary injury
❑Cephalic tetanus: 1o infection is in the head Generalized tetanus:
(ear); involvement of cranial nerves Opisthotonos (back
spasm)
❑Neonatal tetanus (tetanus neonatorum):

generalized disease in neonates associated with
an initial infection of the umbilical stump
Neonetal tetanus (tetanus

neonatorum)
64
Laboratory diagnosis C. tetani
❑ C. tetani: 1o diagnosis rests on the clinical picture
❑ Specimens: are usually wound swabs, or excised bits of tissue

from necrotic depth of wound
❑ Microscopy: – drum stick or tennis racquet appearance
❑Culture: C. tetani is a strict anaerobe with a temperature

range of 14–43 ºC (37 ºC optimum).
65
C. tetani……..
❑Robertson’s cooked meat medium (RCMM): It is also

known as cooked meat broth (CMB)
▪ Culture in RCMM, C. tetani is slowly proteolytic
▪ Culture in cooked meat broth produce blackening of meat
with foul smelling product.
▪ If Clostridia growth occurs (check a Gram smear), divide
the culture and heat one half at 80 ºC for 30 minutes and
cool
▪ Subculture both the unheated and heated cultures on
fresh blood agar and incubate anaerobically.
66
C. tetani……..
❑Blood agar: On fresh blood agar, C. tetani produce 

hemolysis, later develops into - hemolysis due to the
production of hemolysin (tetanolysin)
❖When isolated (only very occasionally), C. tetani produces

a fine film of feathery growth.
• Use a hand lens to examine the plate.
67
C. tetani……..
Antitoxin test
▪ Proof of isolation of C. tetani must rest on production of toxin
and its neutralization by specific antitoxin.
▪ If growth occurs on blood agar, subculture on a blood agar with
antitoxin plate (half the plate covered with antitoxin).
▪ Incubate the plate anaerobically.
▪ The haemolysis produced by C. tetani is inhibited by the
antitoxin.
68
Prevention & control
▪ Passive/Active immunization (antitoxin globulin),
▪ Vaccination: Tetanus toxoid given alone or with DPT

vaccine
▪ Proper care of wounds contaminated with soil
▪ Prophylactic use of antitoxin
▪ Rx: proper wound care and use of antitoxin and penicillin,

chloramphenicol, metronidazole, imipenem
69
Gram negative rods
70
Introduction
❑ Gram negative rods comprises:

◼Enteric Gram-Negative Rods
(Enterobacteriaceae)
◼Pseudomonas
◼Vibrio
◼Campylobacter
71
I. Enterobacteriaceae
❑Non- spore forming, aerobic and facultative anaerobic Gram
negative rods
❑They are found as normal flora primarily in colon of humans

and animals
❑Salmonellae and Shigella are pathogenic for humans
❑Also named as coliforms or enterobacilli
❑Grow over a wide range of temp. in ordinary media.
❑Most are motile
72
General characteristics….
❑These families of bacteria are of importance to food

manufacturers as many members of this family are a normal
gut ﬂora of human and animals.
❑Enterobacteriaceae are also found in water or soil and as
such are used as key indicator organisms to determine the
presence of more virulent and pathogenic bacteria.
73
General characteristics….
❑All ferment glucose with acid production
❑They are catalase-positive, oxidase-negative, and reduce

nitrate to nitrite
❑They possess a complex antigenic structure, and produce a

variety of toxins and other virulence factors
❑Release endotoxin from their cell wall
❑Some release exotoxin
74
Enterobacteriaceae …
❑Most of the genera possess three type of antigens

—H- antigen- flagellar protein and heat labile (destroyed at 60-100 ºC).
—K-antigen - Capsular polysaccharide or protein, surround the cell wall.
—O (somatic) antigen - found in the cell wall and are heat stable.
❑The family includes many genera, such as;
▪ Escherichia
▪ Shigella
▪ Salmonella
▪ Enterobacter
▪ Klebsiella, Serratia, Proteus, and others
75
Classification of Enterobacteriaceae
❑Enterobacteriaceae are consists of 40 genera,150 species

and most infections are caused with 20 species
❑Based on lactose fermentation on growing medium:-
Lactose fermenter Non- lactose fermenter

▪ Escherichia species ▪ Salmonella species
▪ Klebsiella species ▪ Shigella species
▪ Enterobacter species ▪ Proteus species
▪ Citrobacter species ▪ Yersinia species
▪ Serratia species ▪ Serratia,Providencia, Morganella….
76
Genus Escherichia
❑The genus Escherichia consisting of five species, of which

Escherichia coli is the most common clinically important
❑Escherichia coli has the following characteristics:
—Most abundant normal flora in human and animal GIT
—Found in soil, water and vegetation
—Because of this association, E. coli is used as an indicator of
fecal pollution of drinking water.
—Is also used as an indicator of fecal contamination of any
food product.
—Most are motile; some are capsulated
77
Escherichia coli…….
❑Virulence factors: Pilli, capsule, endotoxin (LPS), exotoxin,

hemolysins, siderophores, flagella
❑It will not grow at a minimum temperature of 8°C, in a

maximum salt concn of 8%, and a minimum pH of 5.6.
78
Clinical features
❑Cause extraintestinal disease, such as;

— Urinary tract infection (due to uropathogenic E. coli)- limited to bladder
(cystitis) or can spread to kidneys (pyelonephritis) or prostate (prostatitis)
— Wound infection- appendicitis, peritonitis, sepsis and endotoxin induced
shock
— Neonatal septicemia and meningitis in children under 5yrs
— Bacteremia: caused by most common gram-negative rod
79
Diarrhea causing strains of E. coli …
❑ More recently it was noticed that E. coli cause four different

syndromes of gastrointestinal disturbance.
❑Transmission occurs through food and water contaminated with
human waste or by person-to-person contact
1. Enterotoxigenic E. coli (ETEC)
▪ Colonization factor of the organism promote adherence to
epithelial cells of small intestine followed by release of enterotoxin
which causes toxin-mediated watery diarrhea in infants and young
adults
▪ It is an important cause of traveler's diarrhea
80
Diarrhea causing strains of E. coli……
2. Enteropathogenic E. coli (EPEC)
▪Causes outbreaks of self-limiting infantile diarrhea,

especially in locations with poor sanitation
▪Causes vomiting, fever and prolonged diarrhea mainly in
under 2 years infants due to adhering of bacteria to
epithelial cells, have no toxins or invasin.
▪Antibiotic treatment shortens the duration of illness and
cure diarrhea
81
3. Enteroinvasive E. coli (EIEC)

▪Non-motile, non-lactose fermenting E. coli invades the
mucosa of the ileum and colon, and cause tissue
destruction and inflammation w/c leads shigellosis-like
dysentery in children in developing countries
▪Characterized by fever and dysentery like diarrhea with
blood, mucus, and many pus cells in faecal specimens
▪The disease is due to bacteria invading and multiplying in
epithelial cells & not toxins.
82
4. Enterohaemorrhagic E. coli ( EHEC)
▪Causes life-threatening hemorrhagic diarrhoea (colitis) in

all ages, without pus cells, and often without fever
▪ It can progress to hemolytic uremic syndrome (HUS), w/c is
characterized by acute renal failure, hemolytic anemia and
low platelet count
▪ Infection occurs by ingesting contaminated meat products,
un pasteurized milk and dairy products
▪ EHEC is due to cytotoxins damaging vascular endothelial
cells, and is mainly associated with the sero-group O157:H7
▪ It is sometimes referred to as VTEC (verocytotoxin-
producing E. coli).
83
5. Enteroaggregative E. coli (EAEC)

▪The bacteria adhere and aggregate to human intestinal
mucosal cells and produce toxins that causes acute and
chronic diarrhea
▪Produce food-borne illness in developing countries
84
Laboratory Diagnosis
▪ Specimen: urine, stool, pus, blood, body fluid

▪ Gram stained Smear: short Gram-negative rods
Gram staining of a urine sediment preparation

85
Culture:
❑Grow best in aerobic and facultative anaerobes at 35-370C
❑On MacConkey agar: E. coli produce red to pink colonies due
to lactose fermentation with acid and gas production
❑On blood agar: Non-haemolytic, but some strains are β-
haemolytic.
❑On Cystine lactose electrolyte deficient agar (CLED) and
Xylose lysine Deoxycholate (XLD): E. coli produce yellow
colonies
❑On Sorbitol MacConkey agar: E. coli produce pink colonies,
except E. coli O157 that form colorless colony b/c it is non
sorbitol fermenter
86
LF and NLF colonies in MacConkey Agar
87
Culture:……
❑On KIA or TSI agar: differential slope media: three reaction takes
place
• Fermentation of glucose and lactose (acid/acid= LF/GF= yellow/yellow,
Base/acid=red/yellow=NLF/GF, Base/Base= red/red=NLF/NGF)
• Productions of H2S (blackening in the tube)
• Gas productions (crack in the tube)
• E. coli on KIA or TSI agar –

• A/AG (i.e. LF/GF= yellow/yellow in tube)
• Gas production, but not hydrogen sulphide
88
Conti……..
 Triple sugar iron (TSI) agar

or Kligler’s Iron agar (KIA)
 Sugar fermentation TSI-
glucose 1X, lactose 10X,
sucrose 10X
 Sugar fermentation KIA -
glucose 1X, lactose 10X
 Iron salts in both detect H2S
 Phenol red indicator -

yellow in acid
89
Biochemical tests-
❑Lactose fermentation = Acid with or with out gas
production
— Lactose positive (Ferment lactose)
— Indole positive
— Citrate negative
— Lysine decarboxylase (LDC) positive.
— Beta-glucuronidase (PGUA) positive (E. coli 0157 is
negative).
— Methyl red-----------------positive
— Voges-Proskaue (VP)---- negative
90
❑Sanitary measures to prevent fecal-oral transmission:

 Hand washing and proper preparation of food
 Chlorination of water supplies
 Sewage treatment and disposal
❑Rx: Parenteral or oral fluid and electrolyte replacement

is used to prevent dehydration
❑Broad-spectrum antibiotics are used in chronic or life-
threatening cases
91
Salmonella Species
❑Gram-negative, flagellated facultative anaerobic bacilli
❑They are motile and intracellular parasite that survive in
phagocytic cells
❑The illness is an infection due to the ingestion of live
Salmonella cells.
❑They are parasites of man, animals and birds.
❑Incubation period
• May be 14 hours
• But may be shorter or longer depending on :
• Number of cells ingested
• Virulence of the serovar
• Natural resistance of the individual.
92
Salmonella….. cont’d…
• There are more than 2,500 serovars that are distinguished
❑Species of medical importance are;

—Salmonella typhi
—Salmonella paratyphi
—Salmonella enteritidis
—Salmonella typhimurium
93
Virulence factors
1. Endotoxin
2. Capsule
3. Adhesions: slime (mucus) or M antigen ; and the
fimbrial / F antigen
4. Salmonella pathogenic islands (SPI) (genes for
virulence)
1. SPI-1:- helps to invade intestinal epithelial cells
2. SPI-2 helps to survive inside macrophages
5. Flagella – help bacteria to move through intestinal
mucous
6. Enterotoxin - may be involved in gastroenteritis—
involved in food poisoning
94
Pathogenesis & Clinical features
❑Salmonella results in three types of infection:
a. Gastroenteritis
b. Enteric fever/ typhoid or paratyphoid fever
c. Septicemia/ systemic infection
1. Enterocolitis (Salmonellosis): resulting from a food borne
infection
—Manifests with initial watery diarrhea, and later bloody mucoid
diarrhea associated with crampy abdominal pain and vomiting
develop within 48 hrs of ingesting contaminated food or water
—Mostly caused by non-typhoidal Salmonella species (S. enteritidis &
S. typhimurium)
—The disseminate from the intestines to cause systemic disease
95
Salmonella cont’d…
❑Symptoms include diarrhea, abdominal cramps, fever,

nausea, and vomiting.
❑Annually there are about 60 outbreaks with 2,500 cases and

10% mortality due to Salmonella infections.
❑The MO gains entry to food from persons recovering from

an illness, but are still shedding the bacillus.
96
Pathogenesis …
2. Enteric fevers: a severe, life-threatening systemic illness,

characterized by fever and, frequently, abdominal symptoms.
❖It is sever typhoid fever caused by S. typhi and mild paratyphoid fever
caused by S. paratyphi, and transmitted by fecal-oral route via
contaminated food and drinks
❖Complications can include intestinal hemorrhage and/or perforation
and, rarely, focal infections and endocarditis.
❖The organism multiply in sub mucosa of payer’s patches and then
spread to the phagocytes to the liver, gallbladder and spleen resulting in
carrier state
3. Septicemia: results in seeding of many organs, with
osteomyelitis, pneumonia, meningitis
97
Pathogenesis of typhoid fever
98
• Specimen: Blood, stool , urine, serum for enteric fever
• Gram stained Smear: Gram -ve rods
▪ Blood -80% positive in the first week.
▪ Stool (gastroenteritis) - 70-80% positive in the second and third week.
▪ Urine- 20% positive in the third and fourth week.
❑ Serological test: Serum widal test measures agglutinating

antibody levels against O(somatic) and H (flagellar) Abs
- positive after the second week of illness
99
Culture:
1. Differential medium- used for rapid isolation of lactose
fermenter from non-fermenter. Example : EMB agar and
MacConkey agar: give rise to small colorless colonies
2. Selective medium- such as Salmonella-Shigella (SS) agar,
XLD agar, Deoxycholate citrate agar favour growth of
salmonella and Shigella over other Enterobacteriaceae.
On XLD agar, Shigella and Salmonella spp. produce small red
colonies, most strains of Salmonella with a black centre.
3. Enrichment cultures: Inhibit normal intestinal flora and
permit replication of salmonella. Example: On Selenite F
broth and Tetrathionate broth Salmonellae are non-lactose
fermenting and some produce H2S.
100
Lab. Dx …Biochemical Rxn
—NLF on MacConkey agar
—On KIA agar Salmonella spps produce
—an alkaline slant, acid butt,
—Produce H2S, except for most strains of S. paratyphi A
—Produce gas from glucose fermentation (except S. typhi)
—Catalase positive
— Urease, Indole and oxidase negative
Biochemical reaction Salmonella Shigella

Citrate + -
Gas + -
H2S + -
Indole - +
Lysine + -
Motility + - 101
Treatment
❑Rx: third generation cephalosporins, azithromycin, and

fluoroquinolones as empiric therapy for typhoid fever while
awaiting the results of antimicrobial susceptibilities
Prevention and Control
▪ Personal hygiene.
▪ Proper storage of food
▪ Use of pasteurized milk and milk products.
▪ Proper cooking of Vegetables and fruits
▪ Health education
102
Shigella Species
❑Gram-negative, non-motile, facultative anaerobic rods
❑The genus is divided into four serogroups with multiple

serotypes:
— S. dysenteriae: cause most severe infections

— S. flexneri: the most common cause of infection in
developing countries
— S. sonnei: common cause of infection in developed
countries
— S. boydii
103
Pathogenesis
❑Cause disease exclusively in the GIT

❑Transmission is from person-person or contaminated
food and water.
❑The incubation period is from 14 hours to a few days.
❑Infection is initiated by ingestion of Shigellae (fecal-oral
contamination)
❑The 5 F’s (fingers, flies, food, fomites & faeces) are the
principal factors in transmission
104
❑It causes shigellosis (bacillary dysentery) characterized by

sudden onset of bloody mucoid diarrhea, abdominal cramp, fever,
generalized muscle ache and weakness
❑Upon ingestion, it will travel to the small intestine where it attaches
itself to epithelial cells, causing ulceration.
❑Symptoms are primarily diarrhea and abdominal cramps.

❑Complications:
 Dehydration
 Electrolyte loss and acid-base disturbance
105
Clinical significance …
❑Outbreaks involving this MO are primarily a problem in

crowded urban institutions.
❑Shigellosis is high prevalent in:

— Poor sanitation areas
— Poor personal hygiene
— Polluted water supply
—Young children are frequently affected
106
❑Specimen: faeces (fresh and mucoid)

❑Microscopy: saline wet preparation shows large no. of
RBC and cells
❑Gram reaction: Gram-negative non-motile rods
• Culture: Non- lactose fermenting colonies on MacConkey, SS agar and Eosin -
methylene Blue agar
107
Biochemical tests:
❑Shigella strains are oxidase-negative, non-motile, lysine-

decarboxylase negative, and urea is not hydrolysed.
❑On KIA they produce an alkaline slant and acid butt, no H2S,
and no gas, except for S. flexneri serotype 6 and S. boydii
serotype 14, which are aerogenic.
❑Catalase is produced except for S. dysenteriae serotype 1,

which is catalase-negative.
108
Prevention, control & treatment
❑Prevention of fecal-oral transmission is the most effective

control strategy
❑Antibiotic treatment of infected individuals
❑The main Rx is fluid and electrolyte replacement
❑Severe dysentery is treated with ampicillin, trimethoprim -

Sulfamethoxazole, or, in patients over 17 years old,
fluoroquinolones such as ciprofloxacin
109
Gram negative curved rods and other
enteric pathogens
The genus
•Vibrio
•Campylobacter
•Helicobacter
•Pseudomonas
The Genus Vibrio
Comma or “S” shaped, gram–negative rods
Actively motile bacteria with single polar flagellum
Facultative anaerobes and prefer alkaline PH
Found in fresh water, shellfish and other sea food.
Readily killed by heat and drying; dies in polluted water but may
survive in clean stagnant water, esp. if alkaline, or sea water.
Most produce oxidase and catalase, and ferment glucose without
producing gas
Vibrio are distinguished from Pseudomonas by being
fermentative as well as oxidative in their metabolism.
111
Medically Important species
V. cholerae & V. parahaemolyticus are clinically

important
Others are V. mimicus and V. vulnificus
Vibrio cholerae
V. cholerae is the most medically important species of
the groups.
Based on Lipopolysacchride O Ag, more than 139
different serogroups have been described.
Vibrio cholerae O1 & O139 cause secretory and non
inflammatory diarrhea
112
Vibrio cholerae…..
❑Causes very severe diarrhea (watery stool), resulting in
dehydration, loss of electrolytes, shock, and death within
days.
❑Symptoms appear within one to two days.
❑Treatment requires antibiotics and the replacement of lost
fluid and electrolytes.
❑The organism is transmitted by polluted water and can be
carried by vegetables and other foods that are not cooked.
❑This bacterium is a G-ve, comma-shaped, motile (single
polar flagellum), aerobic rod.
113
Pathogenesis
❑Transmitted by the fecal-oral route

❑Vibrio species are sensitive to acid, and most die in the
stomach
❑Surviving virulent organisms may adhere to and colonize the
small bowel, where they secrete the potent cholera enterotoxin
(CT, also called "choleragen")
❑CT consists of a numbers of subunits: one A or enzymatic
subunit and five identical B or binding subunits
114
Pathogenesis …
❑The A & B subunits of the toxin binds to intestinal epithelial

cells and causes a rise in cyclic adenosine-monophosphate
(cAMP) production
❑The rising of this cAMP level causes massive secretion of

electrolytes and water into the intestinal lumen result in rice
water diarrhea
115
Characteristics of the disease
▪ Profuse watery diarrhea: rice water diarrhea contain mucus,

colorless, odorless and large numbers of vibrio
▪ Massive loss of fluid and electrolyte with no abdominal pain
▪ Rapidly lose of body weight (>10%)
▪ Dehydration and shock
▪ Death from shock may occur 12 hours or less
▪ Cardiac and renal failure
▪ Sunken eyes, decreased skin turgor
116
Vibrio Parahaemolyticus
❑This is a marine organism, growing well in the presence of

1% to 3% salt.
❑Does not cause nearly as severe an illness as V. cholerae
❑Cause - diarrhea, abdominal cramps, nausea, vomiting, and

to lesser extent headache, chills, fever, and bloody stool.
❑Foods involved include crabmeat, processed lobster, boiled

shrimp, cooked oysters, and raw oysters.
❑Incubation period is 12 - 24 hours
117
❑Specimen: fresh Stool

❑Gram stain: Gram- ve motile curved rods seen using dark-
field microscopy (Motility is best seen using dark-field microscopy)
❑Culture: MacConkey, Blood agar, Thiosulfate-citrate-bile

salts–sucrose (TCBS) medium
❑Grow in alkaline pH aerobically and facultative anaerobes
❑Vibrio strains grow as pale, NLF colonies on MacConkey
agar.
118
Culture…..
❑TCBS agar, selective media for primary isolation of V.

cholerae. On TCBS agar, V. cholerae produce large, sucrose-
fermenting yellow colonies, V. parahaemolyticus produce
Non-sucrose-fermenting green-blue colonies after 18-24 hrs
of incubation
119
Culture…..
❑Alkaline peptone water is selective enrichment media for

V.cholerae-01and used also for transport medium
❑On blood agar sub-cultured from Alkaline peptone water, V.
cholerae produce Beta- hemolytic colonies
120
Biochemical tests:
❑NLF, oxidase positive, ferment sucrose and maltose(acid; no

gas) and do not ferment L-arabinose.
❑On KIA or TSI agar: alkaline/acid= pink/yellow= NLF/GF

without gas and H2S production
❑Serology: polyvalent antisera presumptive diagnosis (Ag
test for O1&O139): Inactivation of vibrio in a wet preparation
after adding vibrio antiserum.
▪ Cholera and Bengal screening test— Agglutination test
▪ Cholera and Bengal smart test— Immunochromatographic test
121
❑ Public health measure that reduce fecal contamination of

water supplies and food
• Adequate cooking of foods can minimize transmission
❑Treatment: replacement of fluid and electrolyte and

antibiotics tetracycline or Doxycycline
122
The Genus Campylobacter
▪ Originally taught be Vibrio, they were latter placed in their
own genus.
▪ They are motile, curved, strictly microaerophilic gram-
negative rods similar in morphology to Vibrio.
▪ The cells have polar flagella and are often are attached at
their ends giving pairs “S” shapes or a “seagull” appearance.
▪ Campylobacter cause both diarrhoeal and systemic diseases.
123
The Genus Campylobacter….Cont’d
❑A total of 16 Campylobacter species are now recognized.

❑Those species of medical importance are;
▪ Campylobacter jejuni (in developed countries)
▪ Campylobacter coli (in developing countries)
❑Of these C. jejuni, and C. coli are the most common and
similar enough to be considered as one.
❑Less medically important Campylobacter spps includes:
▪ C. upsaliensis - C. rectus
▪ C. fetus - C. Lari
▪ C. concisus
124
Pathogenesis and clinical manifestations
❑The jejunum and ileum are the first sites to become colonised, but
the infection extends distally to affect terminal ileum and usually the
colon and rectum.
❑C. jejuni accounts for 90 to 95% of human infections.
❑C. jejuni and C. coli cause gastroenteritis which is characterized by
toxigenic watery diarrhoea or dysentery mainly in children under 2
years.
❑Symptoms include diarrhea, vomiting, abdominal cramps, headache,
and fever.
❑The production of heat-labile enterotoxin resulted in massive cyclic
adenosine-monophosphate (cAMP) production
125
Campylobacter jejuni cont’d…
❑Incubation period is 2 - 5 days, and illness may last one

week.
❑Foods involved include milk, poultry, eggs, pork and beef.
❑The bacterium appears to be spread by food handlers, while

the source may be healthy or sick chickens, cats and dogs.
❑Wild birds are suspected of being the source of C. jejuni in

fresh water.
126
Epidemiology
❑The primary reservoir is in animals and transmitted to humans
by ingestion of contaminated food or by contact with pets.
❑ Commonly found in the normal GIT and genitourinary flora of

animals, including sheep, cattle, chickens, wild birds, and etc.
❑Domestic animals such as dogs may also carry the organisms

and probably play a significant role in transmission to humans.
❑ The most common source of infection is undercooked
poultry, but outbreaks have been caused by contaminated
rural water supplies and unpasteurized milk.
127
Laboratory diagnosis Campylobacter species
❑Specimen: Fresh diarrhoeal or dysenteric specimens

containing blood, pus and mucus
❑Microscopy: Typical appear as simple curved or spiral-

shaped (‘gull-wing or “S”- shaped) gram-negative rods.
▪ Typical darting, tumbling motility of the bacteria under
dark field microscopy or phase contrast microscopy
▪ 1% basic fuchsine staining is presumptive Dx of
Campylobacter from fecal specimen
128
Culture
❑Campylobacter species are strictly microaerophilic, requiring 5–

10% O2 with 10% CO2.
❑C. jejuni and C. coli are thermophilic, i.e. they will grow at 42-43
ºC and 36–37 ºC but not at 25 ºC.
❑ Incubation at 42-43 ºC helps to identify C. jejuni and C. coli from

other species.
❑Improved preston blood-free selective medium, increase

isolations of campylobacter at 37 ºC.
129
Laboratory diagnosis …..
❑On Blood agar: C. jejuni and C. coli produce non-haemolytic

spreading, droplet-like colonies.
❑Selective culture media
▪ Skirrow's Campylobacter selective medium,
▪ Butzller’s Campylobacter selective medium
▪ Preston Campylobacter selective medium and
▪ Blaser's medium,
▪ Campylobacter Charcoal-Based Selective Medium
❑These selective media containing antibiotics such as
vancomycin, polymyxin B, and trimethoprims are selective
culture media for campylobacter species.
130
Biochemical tests
❑A presumptive diagnosis of Campylobacter species can be

made by examinations of the colonies microscopically and
isolating oxidase and catalase positive colonies (from a
selective medium.
Hippurate hydrolysis:
▪ If required, this test (e.g. using a Rosco diagnostic tablet) can
be used to differentiate C. jejuni from C. coli.
▪ Hippurate is hydrolyzed by C. jejuni and not by C. coli.
131
1.Treatment with Erythromycin or ciprofloxacin are drugs of choice

for C. jejuni enterocolitis, but resistance is becoming more
common.
2.Proper cooking of foods.
3.Avoiding contact with infected human or animals and their
excreta.
4.Pasteurizing of milk and milk products.
5.No vaccine available.
132
Summary
Onset of Foods affected and means of
Contaminant
symptoms transmission
Meat and poultry. Contamination occurs
during processing if animal feces contact
Campylobacter 2 to 5 days meat surfaces. Other sources include
unpasteurized milk and contaminated
water.
Home-canned foods with low acidity,
improperly canned commercial foods,
Clostridium botulinum 12 to 72 hours smoked or salted fish, potatoes baked in
aluminum foil, and other foods kept at
warm temperatures for too long.
Meats, stews and gravies. Commonly
Clostridium perfringens 8 to 16 hours spread when serving dishes don't keep food
hot enough or food is chilled too slowly.
Beef contaminated with feces during
slaughter. Spread mainly by undercooked
Escherichia coli (E. coli)
1 to 8 days ground beef. Other sources include
O157:H7
unpasteurized milk and apple cider, alfalfa
sprouts, and contaminated water.
Summary…….
Raw, ready-to-eat produce and shellfish from contaminated
Hepatitis A 28 days
water. Can be spread by an infected food handler.
Hot dogs, luncheon meats, unpasteurized milk and cheeses,
Listeria 9 to 48 hours and unwashed raw produce. Can be spread through
contaminated soil and water.
Noroviruses Raw, ready-to-eat produce and shellfish from contaminated
12 to 48 hours
(Norwalk-like viruses) water. Can be spread by an infected food handler.
Raw, ready-to-eat produce. Can be spread by an infected food
Rotavirus 1 to 3 days
handler.
Raw or contaminated meat, poultry, milk, or egg yolks.
Salmonella 1 to 3 days Survives inadequate cooking. Can be spread by knives, cutting
surfaces or an infected food handler.
Seafood and raw, ready-to-eat produce. Can be spread by an
Shigella 24 to 48 hours
infected food handler.
Meats and prepared salads, cream sauces, and cream-filled
Staphylococcus
1 to 6 hours pastries. Can be spread by hand contact, coughing and
aureus
sneezing.
Raw oysters and raw or undercooked mussels, clams, and
Vibrio vulnificus 1 to 7 days whole scallops. Can be spread through contaminated
seawater.
Prevention of Foodborne Illness
1)Cook- Cook all meat, poultry and eggs to at least

160F. Other than spore-forming bacteria, all
bacteria, parasites and viruses are killed quite easily
with heating to 160F.
2)Avoid Cross-Contamination- Do not cross-
contaminate one food with another. Keep raw food
totally separated from cooked product. Clean
utensils and work areas etc in between working raw
and cooked product. Constantly be thinking of how
microorganisms get from raw to cooked products.
135
Prevention of Foodborne Illnesses
3)Chill Foods- Keep foods cold. After cooking, chill

foods as rapidly as possible. Remember that
cooking has destroyed most of the bacteria but
spore formers, that are resistant to cooking may
become very active and can proliferate rapidly.
4)Cleaning-Wash fruits and vegetables and all foods
possible. In addition, continually wash work areas.
Use only treated or tested water.
136
Prevention of Foodborne Illnesses
5)Personal Hygiene- People working with foods should wash their

hands regularly, wear hairnets, plastic gloves etc. In addition, food
handlers should not work with food if they have a boil, open sores or
feel sick themselves
137
Summary
• Food intoxication & food infection
• A,B and E serogroup of C.botulinum are the primary concern of food industry.
• Clostridium perfiringens and S. aureus are responsible for food intoxication.
• Bacillus cerus causes gastroenteritis due to enterotoxin
• Person with suppressed immune system and including pregnant women are
susceptible to food bone infection by L. monocytogenes
• Food borne infection by S. aureus is a self limited
• Serogroup o1 and non o1 of V. cholera are responsible for food borne

infection by vibirio.
• V. parahemolyticus grow well in the presence of 1-3% salt concentration
138
Other gram negative
enteric rods
The Genus Helicobacter
▪ Microaerophilic, Gram-negative curved or spiral rod
▪ They have a rapid, corkscrew motility resulting from
multiple polar flagella
▪ Distinguished by multiple, sheathed flagella and abundant
Urease productions
▪ Antigenic structure: includes Pili, Protease & Urease
140
Species of medical importance
❑Nearly 22 species Helicobacter are now recognized.

▪ The gastric Helicobacter groups, colonize stomach (The
most common is H. pylori) and the other,
▪ The entero-hepatic group colonize the intestine and liver
(e.g., H. cinaedi, H. fennelliae, H. canis, H.
Heilmanni...etc.)
141
Virulence characteristics
❑Production of urease, cytotoxins and the acid inhibitory protein is

associated with injury to the gastric epithelium layers
▪ The acid inhibitory protein: induces hypochlorhydria during acute
infection by blocking acid secretion by parietal cells
▪ Vaculating toxin (Vac A): has been associated with pore formation in
host cell membranes, the loosening of the tight junctions between
epithelial cells, thus affecting mucosal barrier permeability.
▪ Urease: neutralize gastric acid, stimulate neutrophilia and monocytes
Chemotaxis, stimulate production of inflammatory cytokines
▪ Cytotoxin A (Cag A) : gene is a marker of increased risk of both peptic
ulceration and gastric malignancy.
▪ Flagella and other adhesins: mediate binding to host cells
▪ Catalase: prevent phagocytic killing by neutralizing peroxides
142
Pathogenesis
❑Multiple factors can contribute to the gastric inflammation,

alteration of gastric acid production and others.
❑Initial colonization is facilitated by blockage of acid
production by a bacterial acid – inhibitory protein and
neutralization of gastric acids by the ammonia produced by
bacterial urease activity.
❑The actively motile Helicobacter can then pass through
gastric mucus and adhere to the epithelial cells.
❑Localized tissue damage is mediated by urease by products,
mucinase, phospholipase and the activity of vaculating
cytotoxin that induce epithelial cell damage.
143
Clinical diseases
144
Clinical diseases……
❑In developing countries, H. pylori may also contribute to

diarrhoea, malnutrition and growth failure in young children
(reduced gastric acid protection leads to infection with other
entero pathogens).
145
Pathogenesis……..
• Initial infection with H. pylori causes acute gastritis, sometimes with
diarrhea that lasts about 1 week
• Becomes chronic, with diffuse, superficial gastritis that may be
associated with epigastric discomfort.
• Both duodenal ulcers and gastric ulcers are closely correlated with
infection by H. pylori.
146
Epidemiology
❑Transmission of H. pylori is through ingestion of contaminated food
and drinks
147
❑Specimen: Gastric biopsy, serum, stool

❑Smear: Giemsa or silver stain; H. pylori appears as a small
spiral or S-shaped
❑Histological examination of gastric biopsy a golden method
(Hematoxylin and eosin staining)
❑Used as:-
• Definitive diagnosis of infection
• Determine the degree of inflammation or metaplasia
(abnormal change of cell) and the presence of
lymphoma or other gastric cancers in high risk patients
148
Laboratory diagnosis…….
❑Culture: Isolation of H. pylori may occasionally be required in

the investigation of gastric disease. Using a sterile scalpel and
forceps, cut the biopsy into small pieces.
❑Place a piece of biopsy on Blood and chocolate agar and also in

Christensens urea broth
❑On Skirrow’s media - translucent colonies after 7 days
149
Biochemical reaction:
❑Urease, oxidase, Catalase positive
❑Serology:
▪ Detection of H. pylori specific antibodies in the serum (ELISA)
▪ Detection of H. pylori antigen in stool specimen
▪Special tests: Urea breath test
▪ This non-microbiology test may be performed in specialist
gastroenterology centres.
▪ The patient ingests 13C or 14C radio-labelled urea.
▪ Any carbon dioxide produced by urease producing H. pylori is
detected in the breath using a mass spectrometer
150
❑Improving sanitary hygiene

❑No vaccine
❑Rx: Elimination of H. pylori requires combination therapy
with two or more antibiotics
• A typical regimen includes Amoxicillin + clarithromycin/
metronidazole + Proton pump inhibitors such as
omeprazole or lansoprazole
• Tetracycline + Metrindazole + bismuth salts + omiprazole
151
The Genus Pseudomonas
❑Gram-negative, capsulated, obligate aerobic bacilli and

motile by a single polar flagellum
❑Produce water-soluble pigments
❑Resemble Enterobacteriaceae, but strict aerobic,

oxidase positive and glucose non-fermenter
152
The Genus Pseudomonas……..
❑The most medical important species is : Pseudomonas
aeruginosa
▪ Primarily a nosocomial pathogen and can cause invasive
infection in compromised person
▪ Found in human and animal intestine, water, soil, sewage,
vegetation and moist environment in hospitals
▪ It often produces the non-fluorescent, water and chloroform
soluble bluish pigment pyocyanin, which diffuses into the
agar and colors the pus in a wound blue
▪ Many strains of P. aeruginosa also produce the fluorescent
pigment pyoverdin, which gives a greenish color to the agar
and the pus in a wound
153
Pseudomonas aeruginosa …….
Important properties
▪ Blue-green diffusible pigment
▪ Fruity, grape-like odor
▪ Tends to be very antibiotic resistant
▪ Primarily a water organism but is found as normal GI flora in
about 20 - 25% of people.
Important opportunistic pathogen. Causes many nosocomial

infections, severe infections in neutropenic patients, pneumonia if
cystic fibrosis patients. Serious infections have a high mortality
rate.
154
154
Virulence factors
1. Structural components
▪ Pili: Adhere to epithelial cells
▪ Polysaccharide capsule: Anti-phagocytic property/ inhibit
pulmonary clearance and activity of antibiotics (aminoglycosides)
▪ Lipopolysacchride: Endotoxin effect
155
Virulence factors ........
• Enzyme and toxin production

✓Elastases: Digests protein (elastin, collagen, IgG)
✓Proteases, Hemolysins
✓Phospholipases C (heat labile): Degrade cytoplasmic membrane
components
5. Exotoxin A: Cytotoxic by blocking protein synthesis
156
Virulence factors ........
157
❑P. aeruginosa and causes both localized and systemic illness
▪ Pulmonary infection: range from mild irritation of the bronchi to
necrosis of the lung parenchyma (necrotizing bronchopneumonia)
▪ Primary skin infections (severe burns, wound & soft tissue infection)
▪ UTI in patients with indwelling urinary catheter
▪ External otitis or swimmer's ear
▪ Eye infection
▪ Fingernail infection (frequently exposed to water)
▪ Folliculitis (immersion in contaminated water)
158
Epidemiology
159
❑Specimen: pus, urine, sputum, blood, eye swabs, surface

swabs
❑Smear: Gram-negative rods
❑Culture: Obligate aerobic, grows readily on all routine
media over wide range of temperature (5-42 OC)

❖ Bluish-green pigmented (pyocyanin), large colonies with
characteristic “fruity, grape-like” odor on blood agar
160
Culture:…….
▪ Pseudomonas aeruginosa gives large, flat, spreading pale

colored colonies in MacConkey Agar.
▪ It is oxidase positive and can be identified by its pigments
and/or distinctive smell (characteristics fruity smell).
Figure: The soluble blue pigment

Figure: Pseudomonas
pyocyanin is produced by many, but not
aeruginosa colonies on agar
all, strains of Pseudomonas aeruginosa
161
Culture:…….
162
Biochemical reaction
Important properties:
• Non-lactose and non-glucose fermenter -
ALK/ALK
• Oxidase, Catalase & Citrate positive
• Indole negative
• It does not ferment CHO, instead oxidize
mannitol produce acid
• May be weakly urease positive
163
▪ Care of burnt skin

▪ Proper sterilization of medical equipment
▪ Avoid unnecessary use of antibiotic
▪ Infections are often difficult to eradicate due to P.
aeruginosa being resistant to many antimicrobials.
Treatment:
– Monotherapy is not effective
– Combined therapy is commonly required
e.g. Aminoglycoside and β -lactam antibiotic
164
The end of this Chapter!!!!
165
Gram-negative coccobacilli
The genus
•Haemophilus
•Bordetella
•Brucella
Learning objective:
❑At the end of this chapter the students will be able to:
▪ List the medically-important species of Gram negative
coccobacilli
▪ Describe general characteristics of Gram negative
coccobacilli
▪ Describe the virulent factor of pathogenic species of Gram
negative coccobacilli
▪ Recognize diseases caused by Gram negative coccobacilli
▪ Discuss pathogenicity, clinical manifestations, laboratory
diagnosis, prevention & control of members of the Gram
negative coccobacilli
167
1. The Genus Haemophilus
▪ Haemophilus = “blood loving”
▪ They are fastidious obligate intracellular gram-negative
coccobacilli
▪ Non motile, non-spore forming, variable Catalase rxn
▪ Microaerophilic, require humid enriched environment
▪ Requires medium supplemented with growth stimulating
factor (X and/or V factor)
▪ Causes Respiratory, genitourinary, and CNS infection
168
The Genus Haemophilus…….
❑The main species of medical importance are:

▪ H. influenzae -type b is an important human pathogen
▪ H. parainfluenzae: is associated with otitis media, sinusits
etc.
▪ H. ducreyi: causes STI /soft chancre or chancroid
▪ H. aegyptius: causes acute conjunctivitis
▪ H. aphrophilus: is an uncommon but important cause of
endocarditis
169
Haemophilus influenzae
❑Six serotype based on capsule antigen (a-f)
❑95% of infections caused by H. influenzae type b
❑H. influenzae type b contain poly-ribitol phosphate (PRP)
Capsule –major virulence factor that responsible for serious
systemic infection
❑ H. influenzae requires blood enriched media that contain
hemin (factor X) and NAD+ (factor V) for growth
❑ Other species require only NAD+ (factor V)
❑ Hematin and V factors are both present in erythrocytes
170
Haemophilus influenzae…..Cont’d
171
172
Virulence factor
❑Capsular polysaccharide has anti-phagocytic activity

❑Adherence (fimbiae): contribute in adhesion and invasion
of host tissue
❑Membrane lipooligosaccaride may be responsible in
bacterial attachment, invasiveness, and paralysis of the
ciliated respiratory epithelium
❑IgA protease
• Facilitating attachment to the respiratory mucosa
173
❑H. influenzae type b causes: capsulated strains (invasive)
• Pyogenic (purulent) meningitis in young children < 5
year.
• Leading cause of bacterial meningitis in infants and very young
children (6 months to 1yr)
• Pneumonia and empyema (collection of pus b/n lung and
chest) (mainly adults).
• Acute epiglottitis (acute inflammatory swelling of the
epiglottis and neighboring structures) which may cause
fatal airway obstruction.
• Cellulitis (orbital), Otitis media, sinusitis, septic arthritis,
osteomyelitis, bacteremia, conjunctivitis and occasionally
other invasive infections.
174
Clinical significance……..
❑Non-typable (non capsulated) H. influenzae strains are

mainly responsible for; (Non-invansive)
▪ chronic bronchitis (usually in adults),
▪ Middle ear infections,
▪ paranasal sinusitis
▪ conjunctivitis.
❑H. influenzae is a normal flora of the URT
❑Humans are the only natural hosts, and colonization

begins shortly after birth and infect only human
175
Epiglottitis
17%
Meningitis
50%
Pneumonia
15%
Osteomyelitis
2%
Arthritis
8% Cellulitis Bacteremia
6% 2%
176
Haemophilus ducreyi
• Short, ovoid, Gram negative bacilli, may show bipolar staining.

• They may occur in end to end pairs or short chains in smears
• Require both X but not V factor for growth
Pathogenicity and clinical manifestation
• H. ducreyi causes chancroid, or soft sore.
• It is sexually transmitted and a common cause of genital ulceration.
• The ulcers are painful, shallow and tend to be ragged with marked
swelling and tenderness and it bleed easily.
• Often there is also painful swelling of inguinal lymph nodes, and
abscesses (buboes) may form.
• Chancroid increases the risk of infection with HIV and facilitates
transmission of the virus.
177
178
❑Sample: CSF, synovial fluid, LRT specimen, direct needle

aspiration for sinusitis, otitis, nasopharyngeal swabs, pus,
blood, and spinal fluid for smears and culture
▪ Specimens must be cultured as soon as possible and not refrigerated.
❑Specimens for detection of H. ducreyi should be collected with a
moistened swab from the base or margin of the ulcer
❑Microscopy: gram’s – ve coccobacili (stained with 1% carbon fuchsine)
❑The capsule which surrounds capsulated strains can be

demonstrated by using specific antiserum.
179
Culture:
❑Media must contain iron-containing haemin or porphyrin (as

factor X) and Nicotinamide Adenine Dinucleotide (NAD) or its
phosphate (NADP) (as factor V).
❑Growth is best achieved in a moist carbon dioxide enriched

atmosphere at a temperature of 20–40 ºC with an optimum of
35–37 ºC.
180
Culture:…..
❑On chocolate agar
▪ H. influenzae grows well on chocolate agar because it
contains factors X and V and produce mucoid colonies with
distinctive smell.
❑Heating blood agar to 75 ºC inactivates serum NADase and
releases extra factor V from the red cells.
• Addition of bacitracin (300 mg/litre) provides a selective
medium to recover H. influenzae from sputum.
• This is not needed when culturing CSF.
181
Biochemical test
❑Biochemical test is not routinely done; Satelitism test is

used to identify H. influenzae
Satellitism test
• This is a routine test in identification of H. influenza using S.
aureus that streaked across a plate of sheep Blood Agar and
Nutrient Agar
• After overnight incubation, H. influenzae: shows growth on the
blood agar plate along side of the streak of S. aureus but not on
the nutrient agar plate,
• If satellite colonies are present on both plates the organism is
probably an Haemophilus species that requires only factor V, such
as H. parainfluenzae.
182
❑Active immunization with conjugated polyribitol phosphate

(PRP) given to children younger than age 2 years
❑Currently pentavalent vaccine is available
❑Rifampin is given prophylactically to individuals in close

contact with a patients infected with H. influenzae
❑Treatment : by ceftriaxone
183
II. The Genus Bordetella
❑Extremely small, strict aerobic, non- motile, capsulated,
gram–ve coccobacilli
❑Bordetella species of clinical significance include: B. pertussis
and B. parapertussis
❑Bordetella pertussis, a highly communicable (contagious) and
important pathogen of humans, causes whooping cough
(pertussis) primarily in infants and young children
❑B. parapertussis causes a milder form of whooping cough.
184
Virulence factors
❑Adhesins
• Most important adhesin is filamentous haemagglutinins
• Bind to and agglutinate erythrocytes
• Pili
• Mediate adhesion to ciliated epithelial cells of URT
❑Toxin
• Pertussis toxin (PT) produced only by B. pertussis: Lymphocytosis-
promoting factors- main components of vaccine
• Adenylate cyclase toxin
• Tracheal cytotoxin
• Endotoxin LPS
185
Pathogenicity and Clinical significances
❑B. pertussis causes whooping cough, an infection of the URT
❑Transmission is by droplets and colonize only ciliated cells of
respiratory mucosa and they multiply rapidly
❑Toxin causes the secretion of mucus which leads to irritation
and the spasms of coughing associated with the disease.
❑There is a marked leucocytosis with an absolute lymphocytosis.
❑Complications of infection include lung damage with
empyema, secondary infection leading to bronchopneumonia,
bronchiectasis, convulsions and occasionally brain damage.
186
Clinical stages of the infections
❑After an incubation period of about 2 weeks, the "catarrhal
stage" develops, with mild coughing and sneezing.
▪ At this stage, large numbers of organisms are sprayed in
droplets, and the patient is highly infectious but not very ill.
❑Paroxysmal" stage, the cough develops its explosive character

and the characteristic "whoop" upon inhalation.
▪ This leads to rapid exhaustion and may be associated with vomiting,
cyanosis, and convulsions.
187
Clinical stages of the infections…..
▪ The "whoop" and major complications occur

predominantly in infants;
▪ paroxysmal coughing predominates in older children and
adults.
▪ The white blood count is high (16,000–30,000/ L), with an
absolute lymphocytosis.
❑After 3-4 weeks the disease enters Convalescent stage
▪ Frequency and severity of the coughing gradually
decrease
▪ But secondary complications can occur
188
❑Specimen: saline nasal wash and nasopharyngeal swab

❑Smear: small, capsulated, gram-ve coccobacilli
▪ With toluidine blue stain, bipolar metachromatic granules
can be seen
▪ Organisms are identified by immunofluorescence staining
or by slide agglutination with specific antiserum.
❑Culture – On selective and enrichment medium such as
chocolate-cephalexin blood agar, gray, shiny, mercury like
mucoid colonies at 37 0C for 7 days
189
Culture……..
❑Bordet-Gengou media (Potato-blood-glycerol agar) is

selective media containing a high percentage of blood (20-
30%), to inactivate inhibitors in the agar.
❑Charcoal horse blood agar (Regan-Lowe agar) is the

selective culture media for the proper growth of B.pertussis
Biochemical tests:
▪ Oxidase and Catalase positive
▪ Urease and nitrate -negative
190
❑Sanitary: this is very contagious disease require

quarantine for a period of 4-6 weeks
❑Immunologic: this pertussis vaccine is parts of DPT
schedule
❑Chemotherapeutic: Antibiotics prophylaxis (erythromycin)

may be used for contacts
❑Treatment: of disease with azithromycin
191
The Genus Brucella
❑Gram-negative, non-motile, non- sporulating, aerobic and

short coccobacilli without capsule
❑These bacteria are obligate intracellular parasites that can
survive and multiply within host phagocytes
❑Require specialized media (serum/blood= amino acid,
thiamine, nicotinamide) and prolonged incubation periods for
growth in culture
❑Many require CO2 for growth and Catalase +ve, oxidase +ve
192
The Genus Brucella…….
❑Animals – main reservoir and Brucella are primarily pathogens
of animals, and are associated with particular animal species:
–B. melitensis (goats and sheep, camel)
–B. abortus (cattle, horse, bufallo)
–B. suis (swine/pigs, rodent)
• In additions
— Brucella canis (dogs)
— Brucella ovis (sheep)
193
Virulence factor
❑LPS is the major virulence factor as well as the major cell

wall antigen
• Ability to resist phagocytosis
❑Has certain low-molecular substance on bacterial
surface
• Ability of intracellular survival
194
Pathogenesis and clinical features
❑Brucellosis (undulating fever, Malta fever) is a zoonotic

disease primarily affecting goat, sheep, cattle, buffalo,
pigs and other animals, which is a chronic, lifelong
infection that cause sterility and abortion
❑B. abortus, B. melitensis, and B.suis causes human
brucellosis
❑Transmitted to human either by direct contact with
infected animal tissue, skin and mucus membrane,
during handling of animals and cultures (unbroken skin), or
consuming contaminated milk and milk products (fresh
milk, cheese, cream) or inhalation of aerosols
195
Brucellosis in Humans
❑Human brucellosis known as Bang's disease, named for

who discovered Brucella
❑Occupational hazard of laboratory personnel, veterinarians,
farm workers, and meat handlers at risk through direct
contact or inhalation
❑ Brucellae are intracellular organisms infecting
reticuloendothelial cells of the spleen, liver, kidneys and
bone marrow.
❑ From these sites, the bacteria pass into the blood, and is
characterized by an acute bacteremic phase followed by a
chronic stage.
196
I. Acute stage: Characterized by flulike fever, malaise, sweating

(especially at night), hepatospleenomegaly, lymphadenopathy
and generalized pains associated with fatigue and depression.
▪ Characterized by fever which may be continuous, intermittent,
undulating or irregular (repeatedly rise then fall)
▪ Urogenital symptoms may occur
▪ Often the patient is anaemic and leukopenic with a relative
lymphocytosis
II. Chronic stage: Untreated infections can become chronic with
musculoskeletal symptoms (back pain, chest pain and arthritis).
197
Laboratory Diagnosis
❑Specimen: Blood, biopsy aspirates (BM, LN), serum

❑In microscopic examination, they do not show bipolar
staining but tiny gram-negative coccobacilli
❑Culture: Brucella species are aerobic can grow on
commonly used media, with B. abortus requiring a carbon
dioxide enriched atmosphere 8-10% CO2 at 37oC for 3-4 wks
▪ Blood culture media or chocolate agar (readily grow)
▪ Trypticase soy medium with or without 5% sheep blood,
▪ Brain heart infusion medium,
▪ Serum dextrose agar
▪ Tryptone soya (tryptic soy) diphasic medium
198
Culture:…….
❑Cultures should be kept for 4 weeks with sub-culturing on

solid agar every few days (2-3days).
❑The organisms are more likely to be isolated from the blood

in acute brucellosis during times of fever.
• Isolation is extremely rare in chronic brucellosis
• Brucellae multiply faster in bone marrow cultures, and when
the patient has received antibiotics, the organism are more
likely to be isolated from bone marrow than blood.
199
Culture:…….
❑After a few days of incubation on agar media, the Brucellae

form colonies
▪ They are non-hemolytic.
▪ variety of colonial forms including small, smooth, convex,
mucoid, and rough colonies.
▪ They may be colourless or grey white
Biochemical reaction:
– Catalase, oxidase and urease positive
–Indole negative
–Reduce nitrate to nitrite (Except B. ovis)
200
Laboratory Diagnosis…..
❑Serology: Many cases diagnosed by the standard Plate

agglutination test (Brucella ring test) and an ELISA procedure
for detection of brucella-specific IgM/IgG.
• Drop of serum mixed with drop of Brucella antigen
• Clumping indicates infection
• If the mixture remains clear, the result is negative.
• IgG agglutination titer >1:80 indicate active infection
201
❑Pasteurization of milk and milk products

❑Reduction of occupational hazards
❑Slaughter of all infected animals in dairy herds
❑Vaccination of cattle with live attenuated strain of
B.abortus but is not a 100% effective
❑Treatment:
➢ Treated with combination of tetracycline + doxycycline / Doxycycline + rifampin/
Tetracycline + streptomycin
➢ For infants, tetracycline is toxic, so children are treated with trimethoprim-
sulfamethoxazole.
202
203
Learning Objectives
❑Upon completion of this unit, the student will be able to:

▪ Discuss the basic characteristic of Mycobacterium
▪ Describe the virulent factor, pathogenicity, clinical
manifestations, of Mycobacterium
▪ Discuss laboratory diagnosis, prevention & control of
members of the Mycobacterium
204
The Genus Mycobacterium
▪ Mycobacteria are non motile, obligate aerobe & non -
sporulated long rods
▪ Possess lipid-rich cell walls that poorly stain with Gram
stain.
▪ Does not have the chemical characteristics of either Gram-
positive or Gram-negative bacteria
• Acid-fast bacteria due to their impermeability to certain dyes and stains
• Once stained, acid-fast bacteria will retain primary dye when heated
and treated with decolorizer (3% acid alcohol)
▪ Have slow generation time (15-20 hours) & grow slowly.
▪ Withstand week disinfectants and drying
205
General characteristics……
❑High concentration of lipids/mycolic fatty acids in the cell

wall of Mycobacterium has been associated with:
• Impermeability to stains and dyes
• Resistance to many antibiotics
• Resistance to killing by acidic and alkaline compounds
• Resistance to osmotic lysis via complement deposition
• Resistance to lethal oxidations and survival inside of macrophages
• Prevent attack by cationic proteins, lysozyme and oxygen radicals in the phagocytic
granule
206
Classifications of Mycobacterium
• There are more than 125 Mycobacterium species, including many that
are saprophytes
207
Mycobacterium…….
208
Mycobacterium tuberculosis
❑Non- sporing, non-motile, strictly aerobic, Acid-fast bacilli

❑The main reservoir is an infected human
❑Have slow generation time (18-24 hours)
❑Once stained with primary stain (carbon fuchsine), they
resist decolonization by 3% acid-alcohol
❑Acid-fastness depends on the waxy envelope- mycolic acid

of cell wall
209
Mycobacterium …
❑M. tuberculosis causes tuberculosis and is a very important

pathogen of humans
❑About 1/3rd of the world's population is infected with M.

tuberculosis, with 30 million people having active disease
❑Because of resistance to desiccations, the organisms can

remain viable in the environment for a long time
210
Virulence factors
❑M. tb does not possess the classic bacterial virulence

factors such as toxins, capsules and fimbriae
❑Components of cell wall (mycolic acid, glycolipids,
Peptidoglycan, arabinogalactans/lipoarabinomannon,):
Induce the immediate type of hypersensitivity
▪ Mycolic acid responsible for acid-fastness, granuloma
formation and caseation necrosis
❑Proteins: Elicit tuberculin reaction and antibody
production
211
Transmission
❑Route of infection: Respiratory- Inhalation of droplet nuclei

and ingestion of infected milk
212
Pathogenesis
❑Inhalation of tubercle bacilli *

❑They multiply in the alveolar macrophages * – inhibits
phagosome-lysosome fusion – resists lysosomal enzymes
❑An early tubercle (granuloma) is formed
❑Within 10 days of entry of Bacilli - clones of Antigen specific

T Lymphocytes are produced
❑Can actively produce Cytokines, Interferon ---activate

Macrophages to form cluster or Granuloma.
213
Pathogenesis…….Cont’d
214
The Diﬀerence Between Inactive TB and Active TB Disease
A Person With Inactive TB A Person With Active TB Disease

• Has a small amount of TB germs in their • Has a large amount of active TB germs in
body that are alive but inactive. their body.
• Has no symptoms and does not feel sick. • Has symptoms and feels sick.
• Cannot spread TB germs to others. • May spread TB germs to others.
• Usually has a positive TB blood test or TB • • Usually has a positive TB blood test or TB
skin test indicating TB infection. skin test indicating TB infection.
• Has a normal chest x-ray and a negative • May have an abnormal chest x-ray, or
sputum smear. positive sputum smear or culture.
• Needs treatment for inactive TB to • Needs treatment for active TB disease.
prevent active TB disease.
215
Progression of active tuberculosis
❑Depending on the time of infection TB is classified into;
A. Primary tuberculosis – tubercle formation:
▪ Occurs in a person who has had no previous contact with
the organism
▪ The granulomatous lesions that develops known as tubercle
 About 95%, the infection becomes arrested and 10%
become progressive (active) infections
 The only evidence of tuberculosis
may be a positive tuberculin test
Chest radiograph 216
Progression of active tuberculosis ……
❑If the lesion breaks down, the caseous (cheesy) material

discharged and bacteria may spread to lymph, blood stream,
lungs etc.
❑Progressive disease may lead to;
–Chronic pneumonitis
–Tuberculous meningitis
B. Post primary tuberculosis
 Reactivation of latent infection which is usually seen in
older individual and in those who develop impaired cell
mediated immunity
217
Progression of active tuberculosis ……
218
❑Tuberculosis has two major clinical forms: Pulmonary and
extra-pulmonary tuberculosis
Pulmonary TB (80%)
▪ Primarily occurs during childhood
▪ Secondarily 15-45 years or later
▪ Persistent cough for more than 3 weeks
▪ Productive cough with or without blood-stained sputum
▪ Shortness of breath and chest pain
▪ Intermittent fever, night sweat, loss of weight, loss of
appetite, fatigue and malaise
219
Clinical significance……
Extra pulmonary TB- affects all parts of the body

▪ Most common sites are lymph nodes, pleura, gut, bone
and joints, meninges and peritoneum
▪ TB lymphadenitis
▪ Tuberculosis pleurisy
▪ TB of bones and joints
▪ Tuberculous meningitis
▪ Intestinal TB
220
Risk factors
• Low socieo - economic status

• Genetic disposition
• Chronic infections (HIV/AIDS, Diabetes mellitus, lung
damage)
• More than 10% of all HIV +ve individuals harbor M. tb
• This is 400-times the rate associated with the general public!!
• Previous exposure to Mycabacterial infection
• Malnutrition
221
❑Diagnosis of active pulmonary TB includes clinical symptoms
and abnormal chest radiographs and confirmation by isolation
of M. tuberculosis from relevant clinical material
❑Specimen: Sputum, CSF, pleural and peritoneal fluid
❑Pulmonary tuberculosis: Spot-Morning-Spot specimen of
sputum is collected on two consecutive days (but now Spot-
spot)
222
Laboratory diagnosis….
• In HIV pts the sputum smear may be –ve, because, the bacilli
distributed throughout the body.
• So, EDTA anticoagulated blood Buffy coat smear and pleural

fluid gram smear for lymphocytosis is preferable.
• Microscopic ( ZN or Fluorescent/Auramine stain)
• Direct smear or
• Concentration with NaOH (bleach)
• More sensitive/ positive
• More safer and more digestive sputum
223
Laboratory diagnosis…..
I. Ziehl – Neelsen staining or Acid fast staining: Smear is

made from the specimen on a new glass slide and stained by
the Ziehl-Neelsen technique. It is examined under oil
immersion lens.
▪ Acid fast organisms – stained red
▪ Back ground – blue
224
2. Auramine-phenol fluorochrome technique
❑This technique can be used to detect M. tuberculosis in

sputum, CSF etc.,
❑Rapid (40X objective) and can detect few bacilli present in
specimen when compared to Acid fast staining
❑ The fluorescent stained organisms can be seen glowing

(fluorescing) against a dark background
225
226
Laboratory diagnosis…..
Culture: Need > 6 weeks, but capable of detecting 10-100 bacilli.

• Lowenstein-Jensen (LJ) medium
oIt is the ordinary selective media for tubercle bacilli
oRaised, dry, creamy colonies present after 3-8 wks of
incubation at 370C
oAcid fast bacilli is slow growing, non pigmented
227
• Identification
• Microscopic: Emulsifications on slide
• Mycobacterium colony is hardy to emulsify
• Test for pigmentations: by exposing LJ medium on light –
Non-pigmented and
• niacin production differentiate from other species
• Incubation at 250C—No growth
• Culturing the colony with nitrobenzoic acid– No growth
228
Laboratory diagnosis…….
❑Serology: Serology includes detection of anti-

mycobacterial antibodies in patient serum with various
methods such as ELISA, RIA and latex agglutination
❑ New technologies
• Molecular methods: It is a rapid method based on
DNA amplification directly in clinical specimens
• Routine PCR: Gene amplification
• Real Time PCR: Gene amplification plus quantification
• Sensitivity: 80 - 85%, specificity ~ 99%
• BACTEC (rapid radiometric culture system)
• Bacteriophage(fast plague test)
• Mycodot Ab test against lipoarabino mannon cell wall Ag
• PPD (Purified protein derivatives) test: non specific intra-dermal test used for screening
exposed children
229
230
Treatment
231
Treatment……
Second-line anti-tuberculosis drugs
▪ If 1st line drugs failed: MDR-TB (Isoniazid (INH) & Rifampicin)
• Aminoglycosides (kanamycin, amikacin, capromycin)
• Fluroquinolones (ciprofloxacin)
• Thioamides (ethinoamide, prothionamide)
• Cycloserine
• Paraminosalicylic acid (PAS)
232
Problems with TB treatment
❑Resistance to anti-TB drugs can occur When the drugs

are misused
– Patients do not complete their full course of treatment
– Health-care providers prescribe the wrong treatment, the wrong dose, or
length of time for taking the drugs
– Supply of drugs is not always available

– The drugs are of poor quality
233
Drug Resistance and Tuberculosis
234
Types of drug Resistance
• Mono drug resistance: resistance for one drug among

the five group
• Poly drug resistance: resistance to more than one drug
• Multidrug resistance (MDR): is resistant to at least two

of the best anti-TB drugs, isoniazid and rifampicin
235
235
Types of drug Resistance cont’d
• Extensively Drug-Resistant (XDR): is resistant to

isoniazid and rifampin Plus resistant to any
fluoroquinolones and at least one of three injectable
second-line drugs i.e., Amikacin, Kanamycin, or Capreomycin
236
Prevention
❑Early case detection & treatment

❑Decreasing of over crowding
❑Pasteurization of milk --- ↓M. bovis infection
❑Health education
❑Immunization with BCG vaccine (Bacillus of Calmette-
Guerin) which consists of a live attenuated strain of M. bovis
❑ It is not given for immunocopromized individual and it is
not 100% effective. Studies suggest a 60-80% effective rate
in children
237
237
HIV Counseling
❑All persons with suspected and confirmed TB disease

should be offered HIV counseling and blood testing, in
addition to treatment
❑This is because TB is more likely to occur among HIV

positive individuals
238
Spirochetes
The Genus
•Treponema
•Borellia
•Leptospira
Spirochetes
▪ Helically coiled, spiral or corkscrew shaped gram-negative
bacilli, but stain poorly with gram stain
▪ A characteristics feature is the presence of varying
number of endoflagella
▪ Move by bending and rotating body movements
240
Spirochetes …
❑Depending on the species, they can be microaerophilic,
aerobic, or anaerobic
❑Not yet been cultured
❑Spirochetes of medical importance are
▪ Treponema
▪ Borellia
▪ Leptospira
241
The Genus Treponema
242
Genus Treponema …….
❑Not cultured in artificial media, in fertilized eggs and tissue
culture
❑It is extremely fastidious and fragile and is sensitive to

disinfectants, heat, and drying
❑Actively motile, rotating around their endoflagella
❑Remain viable in the blood or plasma (transmitted via blood

transfusion)
243
Treponema pallidum……
❑It is so thin that it cannot be observed by light microscopy

but requires immunofluorescent or dark field techniques
❑Unlike typical gram-negative bacteria, most spirochetes,

including T. pallidum, do not have Lipopolysacchride (LPS), or
endotoxin, in the outer membrane
❑Antigenic variation of surface proteins plays an important

role in immune evasion
244
Epidemiology
❑Natural infection with T. pallidum is limited to the human host

and incubation period is 3-4 wks
❑Route of transmission is sexual contact or blood transfusion and

transplacentally (congenital syphilis)
❑Sexual exposure to a person who has an active syphilitic chancre

carries a high probability of acquiring syphilis
❑One it enter in the sub-epithelial tissue, the organism replicate

locally and form chancre
245
Clinical features
❑Acquired syphilis has four stages

❑Primary stage: The first symptom of primary syphilis is a
superficial hard, painless genital or oral ulcer (chancre) that
develops at the site of inoculation
▪ T. pallidum: a hard chancre formation (painless)
▪ H. ducreyi cause soft chancre (painful)
❑This primary lesion heals spontaneously after 2-6 weeks, but the
organism continues to spread via the lymph and blood
246
Stages of Syphilis……
❑Secondary stage: develops in 1-3 months after the

primary lesion heals
▪ Chxed by generalized maculopapular rash on any part of
the body, including the palms of the hands and soles of
the feet
▪ There may be syphilitic meningitis, nephritis, periostitis,
hepatitis and retinitis
▪ Primary and secondary syphilis are rich in spirochete from
the site of the lesion and patients are highly infectious
247
248
Stages of Syphilis …….
❑Latent stage: Patients are symptom-free but relapse can

occur and may last 3 to 30 years
oEarly latent stage: Relapse of signs & symptoms occur
within 2 years of developing primary syphilis and
patients are infectious
oLate latent stage: There is no relapse of symptoms and
signs. Patients are not infectious
❑Tertiary stage: Complications include granulomatous lesion
(gumma; lesion of bone and soft tissue), dementia,
blindness, neurosyphillis, cardiovascular syphillis and even
death
249
Figure: Clinical stages of untreated syphilis
250
Congenital syphilis
❑Route of transmission: transparently from mother to

offspring during gestation (after the third month of pregnancy)
❑Out come: Abortion, fetal death, Still birth, Early neonatal
death
❑Organ damage:
Interstitial keratitis
Hutchinson’s teeth
Deafness
251
252
❑ Specimen: Specimen should be collected with care as the

lesions are highly infectious
– Tissue exudates, serous fluid, secretions for 10, 20, early
congenital syphilis
– Blood, CSF, plasma for 20, latent, 30 and late congenital syphilis
❑Dark field microscopy: Slow motile spiral structure
❑Immunofluorescence stain: Direct fluorescent Ab staining
• They are treated with fluorescent tagged anti treponema pallidum
antiserum
• Observe under fluorescent microscope
❑ Silver stain (Silver Impregnation method)
❑Diagnosis by microscopy is done in 10 & 20 stage and
congenital syphilis with superficial lesion
253
Serological tests
❑Serological tests is based on the detection of antibodies and it

fall into two categories:
1. Non- Treponemal tests for screening,
2. Treponemal tests for confirmation
❑Non- Treponema Serological Tests, includes:

– VDRL slide test,
– Rapid plasma reagin (RPR) card test,
– Toluidine red unheated serum test (TRUST)
254
Serological tests ………
❑Treponemal tests for confirmation includes:
–FTA-ABS: Fluorescent treponemal antibody absorption
test
–TPHA test: Treponema pallidium haemagglutination assay
–TPPA: Treponema Pallidum Particle-Agglutination test
255
Prevention and Control
• Treatment of cases and screen contacts

• Practice safe sex with condoms,
• Health education
❑Treatment: Penicillin, Tetracycline, Erythromycin,

CAF
256
The Genus Borrelia
❑The genus Borrelia are relatively large spirochetes
❑Like Treponema, have endoflagella that make them
highly motile and do not produce endotoxin or exotoxins
❑Borrelia species of medical importance includes:
▪ B. recurrentis cause
▪ B. burgdorferi
▪ B. duttoni and B. hermsii
257
Louse-borne borreliosis = Epidemic relapsing fever

❑Epidemic relapsing fever caused by B. recurrentis and usually
acquired from a body louse (Pediculus humanus corporis), that
excretes organisms in its feces (self-inoculation by scratching after
bite by infected louse), so it is not a zoonosis
❑The ability to change surface protein antigens accounts for the

relapsing nature of the disease (i.e., with each relapse, a new
antigenic variant arises)
258
Tick-borne borreliosis = Endemic relapsing fever
❑Endemic relapsing fever is a zoonosis and can be caused by

a variety of Borrelia species, such as B. duttoni, and B.
hermsii and is transmitted by soft-bodied rodent ticks bite
❑Lyme disease (tick born relapsing fever) caused by B.

burgdorferi and transmitted by the bite of a small hard tick
❑Mice and other small rodents serve as primary reservoirs
259
Clinical feature of Lyme disease …….
❑Lyme disease characterized by three stages:

i. Initially phase is characterized by a unique skin lesion
(erythema chronicum migrans (ECM) often described
as bulls eye rash which is periodically reoccur
ii. Second phase: seen in 5-15% of patients with
neurological or cardiac involvement
iii. Third stage involves migrating episodes of non-
destructive, but painful arthritis
260
261
Bulls eye rash
262
❑Specimens: Blood, cerebrospinal fluid
▪ Bacteria are present in high numbers in the blood during febrile
episodes.
Microscopic examination
▪ Borrelia are large spirochaetes, with uneven size coils.
▪ They can be seen in thick (and occasionally thin) blood smears
stained with Giemsa or Field’s stain
▪ Microscopically it is not possible to differentiate the different species
of Borrelia.
Light Microscopy Phase Contrast Microscopy 263

Culture:
Culture: is not used routinely, however, it grow in

Kelly’s medium
Serology test: have been developed for diagnosing relapsing
fever, but antigens for the tests are not generally available
and many show cross reactions with Treponema.
• avoidance of exposure to ticks and lice and on delousing
(cleanliness, insecticides).
264
Rickettsiae Species
• Rickettsiae were originally thought to be viruses because;
▪ Small size,
▪ Obligate intracellular parasite
▪ Poorly stained in gram stains
• Now Rickettsiae is true bacteria due to;
▪ Structure of cell wall is similar with gram –ve bacteria
▪ Contain both RNA and DNA
▪ Multiply with binary fission
▪ Inhibited by antibiotics
▪ Posses enzyme for kerbs cycle and protein synthesis
265
Rickettsia…..
❑Gram negative bacteria, fastidious, obligate intracellular
pathogens
❑The organism stains red in macchiavello’s stain.
❑Grow in yolk sac of embryonated eggs, cell culture and

laboratory animals.
266
Pathogenesis
❑Transmitted to humans by arthropods, such as fleas, ticks,
mites, and lice
❑The organism invade and multiply in the endothelial cells of
small blood vessels- Induced phagocytosis
❑After engulfment it degrades the phagosome membrane by
producing phospholipase leading destruction of infected
endothelial cells
267
Pathogenesis……..
❑Destruction endothelial cells leads to leakage of blood into

tissues (rash) and organ and tissue damage
❑It manifests with fever, headache, malaise, skin rash and

enlargement of liver and spleen
uRickettsial species cause a petechial rash in early disease that

starts on the trunk and spreads outward (centrifugal)
268
Clinical Features
❑The genus Rickettsia has three main groups based on their

antigenic structure
1. Typhus group:
• R. prowazekii: Causes epidemic typhus (louse-borne typhus)
• R. typhi: Causes endemic typhus (murine or flea-borne
typhus)
2. Scrub typhus group: Orientia tsutsugamushi: Causes mite-

borne scrub typhus, also known as Japanese river fever and
Kedani mite disease
269
Clinical Features……..
3. Spotted fever group

▪ Rickettsia rickettsii - cause Rocky Mountain spotted fever
▪ Rickettsia conorii - cause African and Indian tick typhus

▪ Rickettsia akari - cause Rickettsial pox
270
1. Rickettsia prowazeckii
271
Conti….
272
Cont…..
❑Epidemic typhus is common among people with living in

overcrowding and , unsanitary conditions (favor spread of
lice), during famine, wars and natural disasters
273
2. Rickettsia typhi
❑It causes Murine (endemic) typhus or flea-borne typhus
❑Clinical Features: similar but usually milder disease than

that caused by R. prowazekii (louse-borne typhus)
❑It is transmitted to man when bitten by an infected rat-flea

and occurs in those individuals living or working in highly rat-
infested area
❑Reservoir: rodents; vector: flea
274
275
❑Specimen: Serum for serological tests

❑The serological tests to diagnose typhus are:
 Fluorescent antibody test
 Complement fixation test
 Weil-Felix reaction:
Some of the antigen of Proteus strain (OX-19, OX-2,OX-
K) agglutinates with sera from patients with rickettsial
diseases
276
Treatment, Prevention and Control
❑Treatment: Tetracycline and chloramphenicol
❑Prevention and Control
▪ Personal hygiene.
▪ Delousing with insecticide.
▪ Removing of vegetations in which rates and mites live.
▪ Tick repellents.
▪ Prevention of tick bites (protective clothing, insect repellents)
▪ No vaccine
▪ Can’t control the reservoir
277
Summary
278
End of this Chapter!!!
279

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Module- 2

Systemic Bacteriology &

By: Mengistu A. (MSc, Asst Professor in Medical Microbiology)

• At the end of this chapter, students will be able to:

• Discuss pathogenicity, clinical disease manifestations

❑Food-borne illness is also called food poisoning caused by

❑Infectious organisms or their toxins can contaminate food at

❑Cross-contamination — the transfer of harmful organisms

Staphylococci make a very large contribution to man's

❑The genus has at least 30 species

❑ S. aureus is a major pathogen for humans

❑ Pathogenesis of Staphylococci is combined effect of:

• Protein A: inhibits antibody-mediated clearance by binding IgG

▪ Toxic for many cells, Lyse RBCs, damage platelets &vascular

❑Inflammatory disease (Superficial Skin infection):- mainly

❑The source of this bacterium is human carriers, who can

❑The illness is self-limiting within a day or two and not fatal,

❑Severity of the symptoms depends on the amount of toxin

Superficial folliculitis Deep folliculitis Furuncle (infected hair follicle

Carbuncle: Multiple abscesses Staph impetigo Scalded skin syndrome

❑Healthcare-associated MRSA: Highly resistant to

❑New type of MRSA identified– Infection in a person with no

▪ Person to person via hands & skin-to-skin contact

❑Specimens: skin swab, pus, sputum or tracheal aspirate, blood

Gram staining preparation of pus: Gram positive cocci in grapelike

❑On Blood Agar : Staphylococcus spps form slightly raised

❑Mannitol salt Agar (8-10% NaCl): S. aureus grow well and

• Catalase Test: breakdown of H2O2 Catalase

▪The catalase test can also be done on a slide. Remove a

▪Coagulase test: causes plasma to clot by

▪ Proper disinfection and sterilization of medical devices

▪ Contact precautions should be used for all patients with

▪ Increased awareness by healthcare providers

❑ Endospores is oval and centrally located

❑ They are thermophilic, psychrophilic, acidophilic, alkalophilic, halotolerant,

❑ Majority are harmless and saprophytic and widely

❑The pathogenicity of B. anthracis depends on two

❑Toxins produced consists of three proteins: Protective

❑B. anthracis is the principal pathogen of the genus which

❑In humans, the infection acquired by entry of spores

• B. cereus is a pathogen that produces enterotoxin and cause

• Gram positive, aerobic, and spore-forming rod.

• Can be controlled by keeping food colder than 10C or

❑Causing two types of gastrointestinal illness, depending on

❑This bacterium mostly found in: Meats, Vegetables, Sauces

❑Typically, the organisms are introduced into the eye by foreign

❑Specimens – pus or fluid or swabs from cutaneous lesions,

❑On blood agar: the B. anthracis produce non-hemolytic

❑ Mannitol egg-yolk phenol-red polymyxin agar (MYPA) is

❑ On Gelatin stab culture: It rapidly liquefies gelatin stabs.

❑Ferment glucose, maltose, sucrose, trehalose, and dextrine,

❑Positive for Nitrate reduction test, Catalase test, starch

❑ B. cereus, unlike B. anthracis, is motile, non-capsulate, and

❑Protective clothing and gloves for handling potentially

❑Active immunization of domestic animals with live

❑ Vaccination of workers in high risk occupation with anthrax

❑ Most species of clostridia are anaerobic gram-positive

❑ Majority are motile, but C. perfrengens is non-motile

❑ Most species are saprophytes found in soil, water, decaying