AFB &

SELECTED GRAM POSITIVES

BLS 206

GENUS: MYCOBACTERIUM

Classification – -Family Mycobacteriaceae

-1 genus of medical importence = Mycobacteria

-All are slow growing

-All are acid-fast and contain large amounts of lipids in their cell walls

-Tubercle bacilli = M. tuberculosis, M. africanum, and M. bovis

•Mycobacteria other than tubercle bacilli (MOTT) or the atypicals. All other species.

•The Mycobacteria are divided into 4 groups (Runyon groups) based on growth rate and pigmentation:

1. Photochromagens:- are non-pigmented when grown in the dark. - produce photoactivated pigments upon exposure to lighte.g M. kansasii, M. marinum, M. asiaticum, M. simiae

2. Scotochromogens: -Produce deep yellow to orange pigments when grown in light or dark,- The color deepens upon two weeks exposure to light e.g M. gordonae, M. scrofulaceum, M. szulgai, M. xenopi.

3. Nonphotochromogens:-May produce pigment ranging from white to yellow, -The pigment does not intensify upon exposure to light. e.g. M. tuberculosis. M. avium, M. intracellulare, M. terrae, M. ulcerans.

4. Rapid growers:- organisms that form colonies within seven days. eg. M.phlei, M. smegamtis, M. fortuitum, M. chelonei.

Morphology and cultural characteristics

Obligate aerobe, Gram-positive rods

Acid fast

Complex cell wall lipids– include mycolic acids– protects vs. phagolysosomal components

Peptidoglycan, glycolipids– acid-fastness

NB: Always work under biosafety cabinet!

Heating required for stain penetration due to the high lipid content of the cell wall (mycolic acid and waxD).

Several acid fast stains that may be used:

1. Ziehl-Neelsen:-uses heat to get the primary stain of carbol fuchsin to penetrate the cell wall;

- acid alcohol destaining;

- methylene blue as the counterstain.

2. Kinyon: – Uses a higher content of phenol (organic solvent) in the carbol fuchsin primary stain to allow penetration of the stain without the need to apply heat.

- Acid alcohol for destaining and

- ethylene blue as the counterstain.

3. Auramine-rhodamine fluorochrome (a fluorescent stain):-Requires a fluorescsnt microscope.-Stain with auramine-rhodamine for 10 minutes (phenol in the solution allows for penetration)-Destain with acid alcohol-Counterstain with acridine orange-A positive result is a bright yellow fluorescence.

Acid-fast bacilli

Highly contaminated specimens with organic debris and normal flora, should be digested and decontaminated with NaOH.

Most grow on simple media.

For primary isolation complex media should be used – Use of a nonselective, a selective and possibly a liquid media is

recommended.

1. Nonselective -May be egg or agar based. - May include malachite green to suppress growth of contaminating bacteria.

a. Lowenstein-Jensen -egg based;

-Colonies grow in 18-24 days.

b. Middlebrook 7H10 and 7H11 – agar based; - colonies grow in 12-14 days.

2. Selective media: – Consists of one of the nonselective media plus added antimicrobial agents (malachite green, cyclohexamide, and nalidixic acid are often used)

-The colonies of M. tuberculosis on the solid media are rough, dry, granular, nonpigmented to buff colored colonies.

3. Liquid media: - Media usually contains tween 80 and albumin and the organisms will grow faster than on solid media

NB: Most Mycobacteria grow best in 5-10% CO2 and at 35-370 C.

Rate of growth and growth in relation to temperaturePigmentation and photoreactivityFurther biochemical testing includes:

1. Niacin reduction -M. tb. Is nitrate reduction+ and – for catalase at 680 C

2. Tween hydrolysis, 3. Arylsulfatase production, 4. Tellurite reduction, 5. Salt tolerance, and 6. Pyrazinamidase production

Identification

M. tuberculosis culture

Virulence factors. Cord factor :

– A glycolipid, trehalose 6,6’ dimycolate responsible for the serpentine growth (filaments or cords) of M. tb. in which the bacilli grow in close parallel arrangement.

-Is toxic to leukocytes, -antichemotactic, -interfees with mitochondrial function in mice and -plays a role in the development of granulomatous lesions

Iron capturing ability – required for survival inside phagocytes

Sulfolipids prevent phgosome-lysosome fusion so that the organisms are not exposed to lysosomal enzymes (important in intracellular survival)

Pathogenecity

1.M. bovis:• Hosts: cattle- natural host, swine,horses, dogs

and sheep!? Cats also susceptible and may perpetuate bovine disease.

• In cattle- pulmonary d’se with involvement of associated lymph nodes.

• Viscera and bone infections- occur in human• Chickens- resistant.• Rabbits, mice and guinea pigs more

susceptible.

2. M. avium

Chicken most susceptibleOther birds-YesNot all infected chickens show gross lesions.Water fowls – resistantIn swine- disease found in lymph nodes of the head.Cattle refractory but sensitizedSporadic cases in horses, dogs and catsInfection in human- little consequence.

• In human and primates• Cattle sensitized by the human organism• Swine- diseases in lymph nodes of the head• Parrots – susceptible• Dogs- can• Cats – resistant• Chicken-rare• Guinea pigs and mice- very susceptible• Rabitts- susceptible

3. M. tuberculosis

On primary isolation:

– visible growth after up to 8 weeksColonies:

– Buff colour, dry bread crumb-like appearance

– Growth is eugonic (M. bovis = dysgonic)Growth temperature:

– 35-37oC Obligate aerobeHeat-sensitiveSusceptible to alcohol, glutaraldehyde and

formaldehyde.

CULTURE CHARACTERISTICS

Differential characteristics oftuberculle bacilli causing animal/human disease

____________________________________________________Species Atmospheric preference Nitratase TCH Pyrazinamide---------------------------------------------------------------------------------------M. tuberculosis Aerobic + S S

M. bovis Microaerophilic -- R R_______________________________________

TCH = thiophen-2-carboxylic acid hydrazide

S= Sensitive, R= Resistant

THE DISEASE

Not highly contagious:

–transmission with prolonged contact between susceptible and active case

–usually transmitted by airborne droplets, must penetrate deep into respiratory tree

–infection can be via other routes: vingestion => infection through cervical or mesenteric LN

Virulence

– Ability to Survive within Macrophages

TUBERCULIN TEST

Tuberculin: a heat-concentrated filtrate of a

broth in which tubercle bacilli had been grown.Injection of tuberculin into the skin >>

– Large, indurated reactions >>Post-Primary Tuberculosis.

– No induration >> Protective immunityPurified Protein Derivatives (PPD):

– Mantoux Method (Intracutaneous)

– Heaf Method (Spring-loaded gun)

– Tine Tests (Disposable single tests)

LABORATOY DIAGNOSIS

2. Microscopy:

– Ziehl-Neelsen Stain

– Fluorescent dyes

3. Culture:

– Decontamination:

– Lowenstein Jensen medium

4. Nucleic Acid Methods:

FAMILY: BACILLIACEAEHOZA, A. S

1. GENUS: BACILLUS

•Gram +ve bacilli• Aerobic• Spore-Forming

I. Bacillus anthracis

•Causative agent of Anthrax.

Distinctive Properties• Large, Square - ended Rods, Arranged in Chains.• Non-Motile.• Spores:• Capsule:– Purple Stained >> McFadyan's Method(Polychrome Methylene Blue).• Colonies on BA: "Medusa Head Appearance"

Bacillus anthracis

PATHOGENESIS

• Capsule > Invasiveness– D-glutamic acid

• Exotoxin (Plasmid mediated)i. Protective Factor (Antigen).ii. Oedema Factor.iii. Lethal Factor.Blocks the Adenyl Cyclase Pathway >Increases vascular Permeability > Shock

LABORATORY DIAGNOSIS:

• Specimens obtained from:a malignant pustule, sputum, blood.

- Gram stain + fluorescent-antibody stain.- Motility- Capsule formation: Sodium bicarbonate+CO2- String-of-pearls reaction:- Mouse test:- API>> Demonstration of Abs to the organism:

Bicarbonate agar and blood agar plate cultures of Bacillus anthracis

Negative encapsulation: Blood agar and bicarbonate agar plate cultures of Bacillus cereus

• TREATMENT– Penicillin, Ciprofloxacin

• IMMUNIZATION–Animals > Live spore vaccine (Sterne strain)– Workers at Risk of Exposure >Anthrax Vaccine Absorbed (AVA) >>“Alum precipitated toxoid”

II. Bacillus cereus

•Food Poisoning.

• Clinical Syndromes:i. Severe Nausea &Vomiting.

ii. Abdominal Cramps & Diarrhoea.

PATHOGENICITY:>> Due to an Enterotoxin.• Also Causes Disease in Patients with Underlying Disease.

TREATMENT:>> Tetracycline, Erythromycin.

• iii. B. subtilis:

• iv. B. stearothermophilus.

2. GENUS: CLOSTRIDIUM

•Large rods with rounded ends, occur singly in short chains,or as long filaments•Gram +ve bacilli• Anaerobic (some facultative microaerophilic)•Most are motile (except C. Perfrigens) and nonencapsulated• Spore Forming-Spores: can be central, subterminal, or terminally•Fermentative •Catalse -ve

Distinctive properties

Groups of Clostridial spores

1. Subterminal spores Gelatin not hydrolysed- group I e.g C. colinum Gelatin hydrolysed- group II e.g C. sordellii, C.

botulinum, C.novyi, C .perfrigens, C hemolyticum, C. chauvoei, C. septicum.

2. Terminal spores Gelatin not hydrolyzed- group III (not associated

with animal diseases) Gelatin hydrolized- group IV e.g C. tetani

Ink Stain of Sporulating Clostridiumsporesappear clear, vegetative cells dark

•Clostridia are free-living saprophytes in soil•Some spp are found in the GIT•Only few spp (>60) cause disease.

Mode of infection•Ingestion: Black leg (cattle); botulinum (food), enterotoxemia, bacillary hemoglobinuria.

•Wounds: C. tetani, C chauvoei (sheep), C. septicum and other gas gangreen organisms infect wounds.

Distribution

I. Clostridium perfringens

• Nonmotile

• Spores Not Produced in Ordinary Media.

• Aerotolerant Anaerobe.

• 5 Types: A - E

Clostridium perfrigens• Synm: C. welchii.• Disease: enterotoxemia• Occurrence: C. perfrigens type A more

widespread, present in air, soil, dust, manure, water of lakes, streams, and rivers.– Has been isolated from vegetables, milk, cheese,

canned food, fresh meat, shellfish and mollusks.

FOOD POISONING:

• Cl. perfringens Type A >> Enterotoxin.> Acute Abdominal Pain and Diarrhoea.

PATHOGENICITY & CLINICAL INFECTION

•α-Toxin: Acts on lecithin-containing lipoproteincomplexes in the cell membrane.

• Predisposing Factors:i. Trauma with deep and lacerated or crushwounds of muscle Etc.

ii. Require a reduced oxygen tension andreduced oxidation reduction potentialfor growth.

Gram stain of Clostridium perfringens

Exudate smear of Clostridium perfringens

Tissue smear of Clostridium perfringens

DISEASE:• Clostridial myonecrosis.

• Less severe wound infections.

• Food poisoning.

LABORATORY IDENTIFICATION• In Chopped Meat - Glucose Medium:•Colonies are 1-3 mm in diameter, round or slightly irregular, slightly raised, granular, and transparent or transluscent.• On BA:• On Egg Yolk Agar:>> Precipitation (Opalescence).

• Milk Media: Stormy Formation.

• Nagler Reacrion:

Blood agar plate with Cl. Perfringens characteristic double zone of hemolysis

Clostridium chauvoeisynonym: C. feseri

• Disease: blackleg• Wide spread, found in intestine and in normal

tissues• Toxins

– α toxin: hemolysin, necrotoxin– ß toxin: deoxyribonuclease– γ toxin: hyaluronidase– ∆ toxin: hemolysin

Phathogenicity• Ruminats: Blackleg (cattle 4 months to

2yr)- ingestion/endogenous, sheep and goats- wounds

• lession dry, dark, with gas bubles, and a rancid odor, there may be bacteremia.

• Immunity: – Formalized whole-broth cultures- life long – Recovery from disease—renders the animal

immune for life

Clostridium septicumDisease: Malignant oedemaOccurnce:Ww, in soil and intestineToxins: 1. α toxin: lethal, lecithinase, necrotizing and

hemolytic2. ß toxin: a deoxyribonuclease and leukocidal3. gamma toxin:hyaluronidase4. Delta toxin: a hemolyzing and necrotizing.

Pathogenicity: as for gangrene caused by C. chauvoei

• affects horses, cattle, sheep, pigs.

Clostridium hemolyticumSynm:C. novyi, type DDisease: bacillary hemoglobinuriaOccurrence:Ww, especially where liver fluke occur??Subclinical infections may occur in some animals (serves as carriers) sheding the organisms via the intestinal tract.Toxins: ß toxin, phosphplipase C, which is lethal, necrotizing, and cause lysis of erythrocytesPathogenicity: infection limited o cattle, and sheep.

LABORATORY DIAGNOSIS:

• Important: Diagnosis of Clostridium Myonecrosis Should Be Rapid and Made on Clinical Grounds.

i. Direct Smear and Gram Stain of Material from Deep Within the Wound.

ii. Culture:Tissue Aspirates or Deep Swabs Taken from Affected Muscle.

TREATMENT:• Clostridium myonecrosis:i. Surgical removal of all infected and necrotic

tissue.

ii. Antibiotic and Antitoxin therapy.

iii. Adminstration of hyperbaric oxygen.

Clostridia that may be associated with gas gangrene:

• Cl. perfringens Type A

• Cl. Septicum

• Cl. novyi Type A

• Cl. Histolyticum

• Cl. Sordellii

II. Clostridium tetani

Tetanus.

> Terminal Spores with drumstick appearance.

• Obligate anaerobe.

•Gram positive rods

Clostridium tetani

VIRULENCE FACTORS:• Tetanus Toxin (Tetanospasmin) > Neurotoxin.i. An Intercellular Toxin Released by Cellular

Autolysis.ii. Inhibits the Release of Inhibitory Transmitters.iii. Toxoid.•Hemolysin (tetanolysin or cytotoxin)•Nonspasmogenic toxin•Horses and human are more susceptible to tetanus

CLINICAL INFECTION & PATHOGENESIS• "Tetanus is Generalized in Nature".• Spores germinate in dirty and neglected wounds with some necrosis•Toxin is elaborated after spore germination.•Predisposing factors:

• Docking and castration wounds, umbilical infections (tetanus neonatorum), parturition (puperperal tetanus), and dehorning.

Immunity: •Totally antitoxic•Strains with different heat-stable and heat labile antigens and 10 serotypes present based on flagellar antigens.

LABORATORY DIAGNOSIS:• > Diagnosis on clinical grounds.

TREATMENT:• Antitoxin- applied prophylactically??•Toxoid- widely used in horses.•Debridement of wound and removal of any foreign bodies.•Pencillin >In large doses.•Mild Tetanospasm: >> Barbiturates.

III. Clostridium botulinum

• > Botulism.• > Gram +ve, spore forming bacilli.• > Strict anaerobe.

•Gram stain of Cl. botulinum, characteristic long rods

A photomicrograph of Clostridium botulinum type A

Blood Agar plate with C. botulinum

VIRULENCE FACTORS

• Botulinum Toxin >>> Neurotoxin.– Serologically 8 types of Toxins >>A, B, C1, C2, D, E, F & G.> Affect the Cholinergic System > Blocks the Release of Acetylcholine (at Points in Peripheral Nervous System).

DISEASE IN HUMANS1. Food – borne botulism:Incubation period: 12-36 Hours to 8 days.

2. Infant botulism:

LABORATORY DIAGNOSISi. Diagnosis made clinically.

ii. Detection of organism or its toxin in the suspected food

iii. Samples of stool or vomit

TREATMENT & PREVENTION

Important: Specific Treatment should begin as quick as possible.>Polyvalent Antitoxin >>> Immediately.>Physiological support >NEVER Use a swollen or defective can.