Professional Documents
Culture Documents
MICROBIOLOGY
BY:
GHADIR SAIED EL-HOUSSEINY
ghadir1981@yahoo.com
Bacillus species
Saprophytic Pathogenic
(Anthracoid bacilli) Bacillus anthracis
Bacillus subtilis
Bacillus cereus
May cause disease
Bacillus thuringiensis
Morphology: Gram positive bacilli arranged in chains;
Bacillus anthracis
• Morphology:
Gram positive bacilli,
arranged in long chains,
non motile,
sporulated in vitro (central)
and capsulated in vivo.
Bacillus anthracis
• Disease: Anthrax (Splenic fever). 3 different forms.
• Pathogenesis
• disease of herbivores (goats, sheep, cattle, horses). Spores ingested with vegetation.
• Humans become infected by contact with infected animals or their products, acquired by the entry of spores
through
• injured skin: cutaneous anthrax (malignant pustule) 95% of cases: in farmers
• ingestion: gastrointestinal anthrax: rare
• by inhalation of spores into the lung: inhalation anthrax (Wool sorter disease OR pneumonic
anthrax): 5%.
• May lead to anthrax meningitis.
• Virulence Factors
• Capsule: The poly-D-glutamic acid capsule (polypeptide) is antiphagocytic. B anthracis isolates that do not
produce a capsule are not virulent
• Anthrax toxins
• Diagnosis:
• Specimen:
- Exudate or pus cutaneous anthrax
- sputum inhalation anthrax
- Stool intestinal anthrax
- Blood, csf anthrax meningitis
- pus, blood dead animals
1. Microscopic examination: stained smears show Gram positive bacilli, arranged in long chains
2. Cultural Characteristics
• Ordinary media eg. Nutrient agar : strictly aerobes, optimum temp. 37oC. Colonies are circular with rough granular surface and
irregular fimbriated edge with medusa head appearance (“curled hair” outgrowth) under low power microscope.
• blood agar: non hemolytic colonies, medusa head appearance
• Gelatin stabs: They liquefy gelatin and growth resemble “ inverted fir tree”
• Semisolid agar:
a. Motile (by swarming); B. cereus
b. Non motile: Bacillus anthracis
• PLET media (polymyxin, lysozyme, EDTA and thallous acetate agar)
The best selective media since it inhibits most contaminating organisms and closely related spore-formers.
Colonies appear creamy white on light amber media.
3. Animal inoculation: injection of guinea pig or mouse with isolated culture, animal die in 2 days.
4. Serological tests
A. Ascoli’s thermoprecipitation test (to demonstrate the antigen)
1. Tissues are boiled in acidified saline , cooled and filtered
2. An aliquot of anti-anthrax serum is put in a narrow tube and an equal volume of the filtrate carefully run on its
top.
3. In positive cases, white precipitate forms at the junction between the two fluids within 1-2 hours at room
temperature.
B. ELISA or hemagglutination assay
Antibodies can be demonstrated in the serum of infected person.
• Prevention and Control
1. Protective gloves for handling infected animals 2. Animal is buried , covered with lime 3. Autoclaving of animal products
4. Active immunization of domestic animals with live attenuated vaccines
a. Pasteur’s vaccine: given in 2 doses with 10 days intervals: attenuated culture by growing B. anthracis at 42.5 C for 24 days.
b. Sterne’s vaccine: live attenuated animal vaccine, single dose , Prepared from non capsulated avirulent bacilli (sterne’s strain), more
stable and safer than Pasteur’s vaccine .
5. Persons with high occupational risk should be immunized:
Anthrax Vaccine Adsorbed (AVA)(trade name BioThrax): An american FDA vaccine for at-risk adults before exposure to anthrax.
Produced from culture filtrates of an avirulent, noncapsulated mutant of the B. anthracis. (contains antigen adsorbed to aluminum
hydroxide)
6. Antibiotics can prevent anthrax from developing in people who have been exposed but have not developed symptoms. eg.
Ciprofloxacin and doxycycline (60-day course of antibiotics).
• Treatment:
• large doses of IV and oral antibiotics : fluoroquinolones (ciprofloxacin), doxycycline, erythromycin, penicillin.
• In possible cases of inhalation anthrax,
• Antitoxins: monoclonal antibodies such as raxibacumab (brand name ABthrax) or Obiltoxaximab (FDA approved)
Anthracoid Bacilli
B. cereus
• Morphology: motile, non capsulated, sporeforming.
• Disease: food poisoning: 2 types:
• Emetic type: occurs 1-5 h after meal (eg. starch-containing foods like fried rice), nausea, vomiting, due to
certain serotypes
• Diarrheal type: occurs 8-16 h after a meal (eg. Improperly cooked meat or vegetable-containing foods) ,
diarrhea and abdominal pain, due to different serotypes
• Pathogenesis: due to survival of the bacterial spores when food is improperly cooked, improperly refrigerated,
allowing the spores to germinate. Bacterial growth results in production of an enterotoxin.
• Treatment: Supportive measures: hydration, electrolyte balance and antibiotics: clindamycin, vancomycin,
gentamicin (resistant to beta lactam ab)
• Diagnosis:
• Specimen:
- Emetic: food remnants
- Diarrheal : stool, food remnants
1. Microscopic examination: stained smears show Gram positive bacilli, arranged in chains, motile, non
capsulated
2. Cultural Characteristics:
blood agar: strongly β hemolytic, no medusa head appearance
Gelatin stabs: They liquefy gelatin not typical “ inverted fir tree”
Semisolid agar: Motile (by swarming)
Bacillus anthracis Anthracoids
Morphology: Gram positive rods, club shape, non motile and non spore forming,
Corynebacterium diphtheria
• Morphology
- Gram positive rod,
- non sporulated, non motile,
- club shaped , arranged in Chinese letter appearance
- Bacilli are beaded (metachromatic or volutin granules) stained by methylene blue or Neisser stain.
• Disease: Conjunctival or skin (Cutaneous) diphtheria (rare caused by non toxigenic strains) transmitted
by contacts.
• Tonsillar (respiratory) diphtheria (most common) transmitted by droplets
• mild fever, sore throat
• Organism grows on mucous membrane with production of exotoxin causing necrosis of
superficial epithelial layer giving grayish white pseudomembrane on tonsils, pharynx,
larynx. cervical lymph nodes are enlarged.
• Further exotoxin production may diffuse to blood stream with toxaemia and affect heart
(heart failure), kidney (kidney failure), and nervous tissue (paralysis) and leading to
death.
1. Microscopic: Staining of direct smears with Gram stain and methylene blue to reveal the
typical metachromatic granules.
2. Culture:
- ordinary media (nutrient agar) at 37 C
- Loeffler’s serum medium (enriched) grow better, white colonies that may turn yellow.
- Blood tellurite media : (blood agar + 0.04% K tellurite) (selective, differential)
- 3 biotypes, All colonies appear as gray /black colonies, but with different sizes and surfaces
• Gravis : causes severe disease
• Mitis: low severity
• Intermedius: intermediate severity
3. Virulence tests (Test for Toxigenicity) should be done to confirm the suspected organism
a. In vivo
- Unknown strain is injected in each of two guinea pigs, one of which is injected 24 hours earlier with diphtheria
antitoxin (control ).
-If the control animal survive while the test animal dies, the test strain is toxigenic
- If both animals survive, the strain is non-toxigenic.
b. In-vitro (Elek’s test)
• B. Passive Immunization:
• Antitoxic serum (5,000 -10,000 units) to contacts of a case.
•Treatment
I. Antitoxic serum (20,000-100,000 units) injected IM or IV after suitable precautions
• Antitoxic serum is produced in animals ( e.g. horse) by repeated injection of toxoid, therefore it may result in
hypersensitivity e.g. anaphylactic shock or serum sickness.
• To avoid such complications:
1. Test for sensitivity to horse serum must be performed before treatment with antitoxin
2. To guard against development of anaphylactic shock, pretreatment with cortisone and antihistaminic is recommended.
3. Epinephrine injection should be ready for use.
II. Chemotherapy: Antibiotics are used in patients or carriers to arrest toxin production
• Metronidazole, Erythromycin (orally) for 14 days, or Procaine penicillin G (IM) for 14 days.
• Patients with allergies to penicillin G or erythromycin can use rifampin or clindamycin.
• They can be used in combination with the antitoxin treatment (but not as a substitute for it)
• lymph nodes in the neck may swell. obstruction in the throat may require intubation or a tracheotomy.
Diphtheroids
• Occur as commensals in man, some produce diseases in animals,
• produce no toxin, non pathogenic.
• Arranged in parallel rows
• These include:
• C. hofmannii : commensals of throat
• C. xerosis : commensal in conjunctival sac
• C. acne: now called Propionibacterium acnes involved in acne and is often present with Staphylococci.
• C. ulcerans: Previously thought to simply be “diphtheroids” are now believed to cause diphtheria.
Listeria sp.
Listeria monocytogenes
• Morphology
• peritrichously flagellated, Gram-positive rods, which are sometimes arranged in short chains, non spore
forming
• General characteristics:
1-Growth at low temperatures (4 C) : making it an important foodborne pathogen
2-Motility at 22–28°C but not at 37°C
3- Virulence factors
two hemolysins (listerolysins)
phospholipases.
• Disease: Listeriosis
• The principle vehicle of infection is contaminated food. (found in both soil and water, cultivated foods like vegetables can
easily become contaminated, unpasteurized dairy or raw foods ).
• Persons at increased risk: pregnant women, neonates, elderly and those with immunosuppressive conditions.
• Symptoms:
• Neonates: develop granulomatosis infantiseptica, a severe disorder involving the internal organs and skin
The neonatal listeriosis has two forms:
• Early-onset listeriosis(within 6 days of birth): is the result of infection in uterus. infant suffers from respiratory
distress, apnea, cyanosis and pneumonia.
• Late-onset listeriosis (7–28 days after birth): infection during delivery; meningitis mainly, the mortality rate 10 -
20 %.
• Pregnant women: mild, flu-like illness, miscarriage, stillbirth, premature delivery, or life-threatening infection of the
newborn.
• eldery and immunocompromised conditions: fever, muscle aches, headache, stiff neck, confusion, loss of balance,
and convulsions. Meningoencephalitis and bacteremia (invasive illness)
• Previously healthy persons: non-invasive illness (meaning that the bacteria have not spread into their blood stream
or other body sites), diarrhea and fever.
• Diagnosis (Early diagnosis of listeriosis in pregnant women is needed to prevent neonatal infection).
• Prevention
1-Thorough cooking of food from animal and vegetable sources.
2-Avoiding consumption of raw unpasteurized milk.
3-Washing of hands, knives etc after handling uncooked food.
Clostridia
• Their natural habitat is the soil or the intestinal tract of animals and humans, where they
live as saprophytes.
• There are 4 medically important pathogenic Clostridium species:
1- Clostridium tetani
2- Clostridia causing gas gangrene: e.g. C. perfringens
3- Clostridium botulinum
4- Clostridium difficile
• Morphology:
All clostridia are anaerobic, spore forming, motile non caps, Gram positive rods .
Clostridium tetani
• Morphology:
-Gram positive bacilli
-swollen at one end due to terminal spherical projecting spores (drum-stick
appearance)
-motile, non capsulated
• Disease: tetanus (lock jaw)
Infection occurs by contamination of wounds with dust containing spores. Germination
of spores is favored by necrotic tissue and poor blood supply in the wound .
• Pathogenesis:
- Tetanus toxin (tetanospasmin) is an neurotoxin produced by vegetative cells at the
wound site.
- This polypeptide toxin is carried to the CNS, where it block release of inhibitory
mediators (e.g. glycine) at spinal synapses.
- The disease is characterized by violent muscle spasms ;
- lock jaw (Trismus) due to rigid contraction of the jaw muscles, which prevent
the mouth from opening
- Spastic paralysis.
- Death usually results from interference with the mechanics of respiration.
- The mortality rate is very high.
• Diagnosis: clinical picture
- Specimen: wound exudates. specimens should be placed in an oxygen-free transport tube or vial.
1- Microscopic: wound exudates are stained for Gram positive bacilli with drum-stick appearance.
2- Culture: blood agar showing zones of β-hemolysis (complete haemolysis) under anaerobic conditions.
Robertson cooked meat medium: Favors the growth of anaerobes
• Treatment: Antitoxic treatment should be started without waiting for laboratory diagnosis:
• Mild cases:
• tetanus immunoglobulin (TIG), also called tetanus antibodies or tetanus antitoxin. (IM)
• Metronidazole IV for 10 days
• Diazepam oral or IV
• Severe cases will require admission to intensive care. In addition to the measures listed above for mild tetanus:
• Human tetanus immunoglobulin injected intrathecally (increases clinical improvement from 4% to 35%)
• Tracheostomy and mechanical ventilation for 3 to 4 weeks. Tracheostomy is recommended for securing the
airway because the presence of an endotracheal tube is a stimulus for spasm
• Magnesium sulphate, as an intravenous (IV) infusion, to prevent muscle spasm
• Diazepam as a continuous IV infusion
• Prophylaxis :
Active immunization: Alum precipitated toxoid is given in combination with diphtheria toxoid and pertussis (DPT)
in IM injections at the age 2, 4, 6 months.
Clostridia causing gas gangrene
Clostridium perfringens (Cl. welchii)
• Morphology:
-Gram positive bacilli
-spores are oval, subterminal and non projecting
- Motile (but no flagella) , capsulated
• Toxins and enzymes
1- Alpha toxin-lecithinase : which damages cell membranes including those of erythrocytes resulting in hemolysis
because it acts on lecithin ( component of cell membrane).
2- Theta toxin causes cytolysis, hemolysis
3- DNAse, hyaluronidase, collagenase
4- Enterotoxin produced by some strains causing food poisoning
• Disease: 1. Gas gangrene (clostridial myonecrosis)
• The condition occurs in deep lacerated, devitalized wounds (car accidents or war wounds) due to contamination
with soil containing the organism or its spores.
• The decreased blood supply lowers the oxygen tension and favors germination of spores.
• Saccharolytic clostridium: Vegetative cells multiply and ferment sugars producing large amount of gas which
distends the tissues and interferes with their blood supply leading to their death.
• They release alpha toxins and hyaluronidase, favor the spread of infection and cause muscle necrosis
• Proteolytic clostridia digest dead tissues leading to change in colour and foul odour of the wound.
• rapidly progressing necrosis, fever, hemolysis, toxemia, shock, and death
• Other common clostridial species that cause gas gangrene include Clostridium bifermentans, Clostridium
septicum, Clostridium sporogenes and Clostridium histolyticum.
2. Food poisoning.
• following ingestion of contaminated warmed meat dishes.
• Toxin is released in the gut. enterotoxin causes diarrhea after 6-8 hours which lasts for 1-2 days.
• usually without vomiting or fever.
• Diagnosis:
- Specimen: gangrene: wound exudates. Food poisoning: stool
1- Microscopic: same
2- Culture: same
3. Biochemical reactions:
1- Organisms ferment carbohydrates with gas production.
2- Organism causes rapid fermentation of lactose in litmus milk and the gas produced splits the clot ``stormy clot
reaction``.
3- Nagler’s reaction: produce opalescence in egg yolk media due to production of lecithinase (hydrolyzes
phospholipids) which causes a visible precipitate around the colonies.
4. Typically, the symptoms of C. perfingens poisoning are used to diagnose it. However, diagnosis can be made using a
stool culture test, in which the feces is tested for toxins produced by the bacteria.
• Treatment:
1. Gas gangrene:
a. The most important: prompt and extensive surgical debridement of the involved area and excision of all dead
tissue, in which the organisms are prone to grow.
b. Antibiotics: Metronidazole, clindamycin plus Co amoxiclav, meropenen or imipenem have anti anaerobic
activity.
c. Polyvalent antitoxin
2. Food poisoning: only symptomatic care
Prevention no vaccine
1. Clean wounds thoroughly (Remove foreign objects and dead tissue)
2. Give antibiotics IV (LA penicillin) before, during, and after abdominal surgery to prevent infection.
3. Food poisoning: Cook food thoroughly and refrigerate
Clostridium botulinum
• Serological characters :
• There are (8) serotypes , A - H which differ in the antigenicity of the neurotoxin.
• The toxin botulinum acts by blocking the release of acetylcholine at the neuro-muscular
junction resulting in lack of muscle contraction and flaccid paralysis.
• Disease: Botulism
• There are three major types:
• Foodborne botulism from ingestion of contaminated canned meat or fish or home-canned
foods (corn, peppers, olives, peas) that may be improperly treated to kill C. botulinum bacteria and
their spores. Such food provides proper anaerobic conditions for growth and production of exotoxin.
• Wound botulism is due to Clostridium bacteria infecting a wound and releasing the neurotoxin.
• In infant botulism, the baby consumes spores of the bacteria which then grow in the baby's
intestine and release the neurotoxin. Honey can contain botulism spores and should not be fed to
babies less than 1 year of age.
• The toxin has the affinity to the cranial motor nerves causing flaccid or bulbar paralysis and death
results from respiratory or cardiac failure.
• There is no diarrhea, vomiting or fever.
• Diagnosis:
- Specimen: food remnants, stool, for wound: exudate, serum
1- Microscopic: stained and examined for Gram positive bacilli
2- Culture: Robertson cooked meat medium.
3. Animal inoculation: Demonstration of toxin in the food extract ;
injected intraperitoneally into guinea pig. After few hours, the animal
will die with generalized flaccid paralysis.
• Treatment:
1. Rapid administration of polyvalent antitoxin (IV)
trivalent antitoxin (A,B,E) or heptavalent antitoxin
(A,B,C,D,E,F,G).
2. Supportive care including mechanical ventilation
• Prevention: The risk from home-canned foods can be
reduced if the food is boiled for 20 minutes before
consumption.
• Other canned foods that are commercially produced and are
bulging (the can is deformed and looks like it is over-
pressurized) or if abnormal-smelling foods are found, they
should be discarded.
Clostridium difficile
• Morphology:
- Gram positive bacilli,
- motile, non capsulated.
- Spores are oval subterminal.
Clostridium difficile
• Disease: The organism is part of the normal flora of gastrointestinal tract .
1. Antibiotic-Associated Diarrhea
• The administration of antibiotics frequently leads to a mild to moderate form of diarrhea
• less severe than pseudomembranous colitis. Mainly due to exotoxin A.
2. pseudomembranous colitis .
• Pathogenesis:
• Antibiotics suppress drug-sensitive normal flora, allowing Cl. difficile to multiply
and produce exotoxins A and B.
• Exotoxin A is an enterotoxin that causes watery diarrhea .
• Exotoxin B is a cytotoxin that causes damage to the gut mucosa.
• This condition is associated with intense inflammation and the formation of a
pseudomembrane composed of inflammatory debris on the mucosal surface.
• Health care providers in the hospital may pass this bacteria from 1 person to
another.
• Diagnosis:
- Specimen: stool
4. Colonoscopy
• Treatment:
1-The causative antibiotic should be withdrawn.
2. Oral metronidazole 500 mg 3 times daily for 10 days
3- Oral vancomycin should be given 4 times for 10 days (severe cases).
4. Fidaxomicin (200 mg 2 times daily for 10 days).
5- Fluids replaced and therapy with probiotics
Mycobacteria
• Are slender, curved rods, strictly aerobic, non sporeforming, non motile.
• cell wall is composed of: peptidoglycan, branched chain polysaccharides,
proteins, and lipids especially mycolic acids (hydrophobic). These lipids endow
mycobacteria with resistance to dehydration, acids, and alkalis.
• Staining: resistant to staining with basic dyes unless they are applied with heat for
prolonged periods of time. Once stained, however, mycobacteria resist
decolorization. The mycobacterial cell wall is acid-fast (i.e., it retains carbol
fuchsin dye when decolorized with acid-ethanol).
• growth rate is much slower than that of most bacteria
• Mycobacteria can be classified into 3 main groups:
• Morphology:
• straight or slightly curved bacilli about the same size as tubercle bacilli.
• non motile and non spore forming.
• They are less acid fast than tubercle bacilli, so 5% sulphuric acid is employed
for decolorization.
• The bacilli are seen singly or in groups
• Aerobic and obligate intracellular pathogens
• Disease: Leprosy
- transmitted from human to human through prolonged contact, e.g., between exudates of a
leprosy patient’s skin lesions, and the abraded skin of another individual.
• Clinical findings
• The disease is endemic throughout Egypt.
• Leprosy is a chronic granulomatous condition of peripheral nerves and mucocutaneous
tissues, particularly the nasal mucosa. Skin lesions are the first sign.
• If untreated, leprosy can progress and cause permanent damage to the skin, nerves,
limbs. Secondary infections, in turn, can result in tissue loss, causing fingers and toes
to become shortened and deformed.
• The incubation period prolonged, so that clinical disease may develop years after
initial contact with the organism.
• two major types; tuberculoid and lepromatous leprosy.
.
Tuberculoid Leprosy Lepromatous Leprosy