SPORE-FORMING GRAM POSITIVE BACILLI

Bacillus and Clostridium Species Can survive in the environment for many years Most do not cause diseases in humans Pathogenic Bacillus spp: B.anthracis B.cereus Pathogenic Clostridium spp: C.tetani C.perfringens C.botulinum C.difficile Bacillus species Strictly aerobic or facultatively anaerobic, saprophytic Rod-shaped, spore-forming, gram-positive Measures 1 x 3-4 um with square ends Catalase positive Spores are located in the center of the nonmotile bacilli Morphology B.anthracis

Bacillus anthracis Common name : Characteristics G+ large bacilli with square ends Occurs in single or paired bacilli (clinical specimen) or long serpentine chains Spores : Encapsulated , NM Facultative anaerobic Habitat and Transmission Habitat is the soil Transmission occurs by contact with infected animals or inhalation of spores from animal hides, hair or wool. Diseases Cutaneous Anthrax Inoculation of spores through cuts or abrasions of the skin Gelatinous edema Edema Vesicle Pustule (malignant pustule) necrotic ulcer - septicemia Pulmonary Anthrax (Woolsorters Disease/Ragpickers Disease) Acquired by inhalation of airborne spores while handling animals Gastrointestinal Anthrax Acquired by ingestion of spores in contaminated meat Occurs in intestinal mucosa Violent enteritis High Mortality Rate Meningitis Secondary infection Pathogenesis 2 major determinants of virulence: Poly-D-glutamic acid capsule : invasion Serum protection McFadyean reaction Anthrax toxin : toxigenicity

B.thuringiensis

Pathogenesis 2 major determinants of virulence: Poly-D-glutamic acid capsule : invasion Serum protection McFadyean reaction Anthrax toxin : toxigenicity

Anthrax Toxin Factor I : Edema Factor (EF) Inherent adenylate cyclase Factor II : Protective antigen (PA) Binding domain Factor III : Lethal Factor (LF) Lethal effects Combined Effects PA+LF = lethality EF+PA = edema EF+LF = inactive PA+LF+EF = edema, necrosis, lethality

Laboratory Diagnosis Microscopy Culture BAM : large, flat, irregular, with slightly undulate margins, nonhemolytic PLET (Polymixin, Lysozyme, EDTA, Thallous acetate) : large, opaque, irregular with curled margins

B.anthracis

Colonies of Bacillus anthracis on blood agar. Biochemical Tests Gelatin hydrolysis (-), slow liquefaction Starch hydrolysis (+) Nitrite reduction (+) Ferments glucose, maltose, fructose, sucrose, trehalose Tests for anthrax ASCOLI TEST Thermoprecipitation test Involves a piece of spleen or hide of infected animal which will be boiled for 15 mins with 10 ml of NSS then placed in a capillary tube with anti-anthrax serum (+) : precipitation of the area where the 2 liquids meet

Lysis of Bacillus anthracis by the lytic phage gamma

String of Pearl Test Inoculate a single streak of the m/o on MHA then place 10U penicillin disk over the streak then overlay with coverslip. After 3-6 hours, place the coverslip on a slide and examine microscopically. (+) Cells assume a circular form due to swelling. Prevention, Treatment and Control Vaccine Sterne Strain of B.anthracis Antibiotic therapy Penicillin, tetracyclines and fluoroquinones are effective if administered before the onset of lymphatic spread or septicemia, estimated to be about 24 hours Knowledge on zoonoses Bacillus cereus Characteristics Similar to Bacillus anthracis NE, motile Habitat and Transmission Habitat is the soil Spores are found on many foods especially rice and vegetables Transmission is by ingestion of organisms or toxins Disease Food poisoning a.Emetic Form fried rice poisoning Short IP : 1-6 hours Nausea, vomiting, abdominal cramps b.Diarrheal Form Meat and vegetable poisoning Long IP : 10-12 hours Abdominal cramps and diarrhea Pathogenesis Enterotoxin Heat-stable toxin in food associated with spore germination (emetic syndrome) Heat-labile toxin produced after ingestion (diarrheal syndrome) STARCH HYDROLYSIS TEST Starch agar tests the ability of an organism to produce -amylase and oligo-1,6-glucosidase, that hydrolyze starch. Iodine turns blue, purple, or black (depending on the concentration of iodine) in the presence of starch. A clearing around the bacterial growth indicates that the organism has hydrolyzed starch.

Diagnosis CASEASE Test Skim milk agar : test the ability of an organism to produce casease. Casein forms an opaque suspension in milk that makes the milk appear white. When casein is broken down into these component molecules, it is no longer white. If an organism can break down casein, a clear halo will appear around the areas where the organism has grown.

STARCH HYDROLYSIS TEST

Laboratory Diagnosis
CRITERION Motility Capsule Hemolysis Growth at 45C Growth on Phenyl Ethyl Alcohol agar Penicillin susceptibility Gelatin hydrolysis Salicin fermentation Gamma phage test Bacillus cereus Motile NE + + R + (liquefaction) + Bacillus anthracis NM Encapsulated S - (inverted) +

Other Diseases caused by Non-Anthrax Bacillus spp Conjunctivitis Keratitis Iridocyclitis Dacryocystitis Orbital abscess Panopthalmitis

CLOSTRIDIUM SPECIES General Characteristics Morphology: gram +, rod-shaped Spore-Former Fermentative metabolism Oxygen requirement: majority of species are obligate anaerobes; however, some species can grow under aerobic conditions or are aerotolerant Clostridium perfringrens a.Characteristics : G+ large bacilli with blunt ends Spore : centrally or eccentrically located with swollen sporangium NM, Encapsulated Anaerobic

Pathogenesis Enterotoxin CHO fermentation in tissues Enzymes Toxins Laboratory Diagnosis Microscopy BAM : large, usually flat, somewhat rhizoid and centrally raised Double zone of hemolysis

Habitat and Transmission Habitat is the soil and colon of man and animals Transmission is by contamination of wound with soil or feces, or by ingestion of contaminated food Diseases Gas gangrene and anaerobic myonecrosis resulting form wound infections Food poisoning that results from ingestion of food (usually meat and poultry) contaminated with enterotoxin-producing strains

Biochemical Tests NAGLER TEST/LECITHOVITELLINE Reaction Test for lecithinase or phospholipase C Presumptive test for C. perfringens Divide an eggyolk medium (Lombard-Dowell) into 2 equal sections. is swabbed with a loopful of C. perfringens type A antitoxin, allow to dry - no toxin (untreated), streaked with C.perfringens Incubate at 37C anaerobically

Gas gangrene

Naglers Test

Clostridium tetani Characteristics: G+ bacilli Spore : round and terminal within a swollen sporangium Motile, NE Anaerobic Specific flagellar antigens

Lecithinase activity results in the production of an opaque zone of precipitation around the area of growth. This precipitation should not be present on that side of the plate previously inoculated with specific antitoxin. Action on Litmus Milk : Stormy Clot Habitat Soil Transmission Contamination of wound with soil Disease : TETANUS (severe tonic muscle spasm) LOCKJAW or TRISMUS : spasm of the masseter muscle; difficulty opening the jaw RISUS SARDONICUS : sarcastic smile Other Interpretations : a.Wide zone of opalescence, no pearly iridescence : C. perfringens, C. bifermentens, C. sordelli b.Wide zone of opalescence, with pearly iridescence : C. novyii, C. botulinum c.Narrow zone of opalescence, with narrow zone of iridescence : C. sporogenes OPISTHOTONUS : persistent spasms, backward arching (breakage of the spine) NEONATAL TETANUS : accounts for about half of the tetanus deaths in developing countries

Tetanus

Habitat SOIL Transmission Organism/toxin transmitted in improperly preserved food and processed cans of food Disease BOTULISM a.Classical/Food-borne botulism Results form ingestion of preformed toxins in food b.Infant botulism Results from ingestion of spores (associated with contaminated honey) and production of toxin in infants gut) c.Wound botulism Results from spore contamination of wound and production of toxin According to WHO d. Adult infectious botulism It occurs as a result of intestinal colonization with C. botulinum and in vivo toxin production in a manner similar to that of infant botulism. These patients often have a history of abdominal surgery, achlorhydria, Crohns disease or recent antibiotic treatment. The disease may simulate a GuillainBarr Syndrome. e. Inadvertent botulism This has been reported in patients who have been treated with intramuscular injections of botulinum toxin. Marked clinical weakness is observed as well as electrophysiologic abnormalities. Pathogenesis 7 toxigenic types : A, B, E, C1, D, E, F, G BOTULINUM TOXIN (neurotoxin) Acts at neuromuscular junctions Inhibits release of excitatory or neurotransmitter acetylcholine Results from spore contamination of wound and production of toxin

Lock Jaw

PATHOGENESIS TETANOSPASMIN (neurotoxin) Attaches to the peripheral nerves, binds to ganglioside receptors, inhibiting the release of inhibitory neurotransmitter glycine results into muscle spastic and death from respiratory failure LABORATORY DIAGNOSIS Microscopy Culture BAM : round, small with fimbraited margins -hemolytic Biochemical assacharolytic Clostridium botulinum Canned good bacillus Characteristics G+ bacilli Spore : oval and subterminal with swollen sporangium Motile, NE anaerobic

Block of transmitter release 1) binding to receptors on the plasma membrane, 2) penetration of the plasma membrane by receptor-mediated endocytosis, 3) penetration of the endosome membrane by pH-induced translocation and 4) intracellular expression of an enzymatic action that culminates in blockade of exocytosis INFANT BOTULISM 5-20 weeks floppy baby SIDS Lab diagnosis : detection of toxin in food or serum of patient Prevention, Treatment and Control Cook food very well Food containers that bulge may contain gas produced by C. botulinum and should not be opened or tasted Adequate ventilation must be maintained in intoxicated patients Clostridium difficile Characteristics: G+ slender bacilli Sporeformer, motile, NE Anaerobic Habitat and Transmission: Normal flora of the gut Transmission by fecal oral route Clostridium difficile on Blood Agar

Diseases: Antibiotic Associated Diarrhrea Disturbance of normal flora (colon, antibiotic) Pseudomembranous colitis : yellow-white plaques on the colonic mucosa Pathogenesis Toxin-mediated damage to the gut wall Laboratory Diagnosis CCFA (cycloserine, cefoxitin, fructose agar) HORSE MANURE-LIKE ODOR

Principle: Milk is an excellent medium for the growth of microorganisms because it contains the milk protein casein, the milk sugar lactose, vitamins, minerals and water. Litmus, a pH indicator is incorporated in the medium for the detection of production of acid or alkali and oxidation-reduction activities. A variety of different chemical changes occur in milk, depending upon which milk ingredients are utilized by the bacteria and is dependent upon the types of enzymes that the organism is able to produce. Litmus milk medium consists of 10% powdered skim milk and the dye molecule litmus. Litmus, upon addition to rehydrated skim milk, changes the colloidal milk suspension from white to lavender (pale bluish purple). Characteristic reactions observed with litmus milk are: 1. 2. 3. 4. 5. 6. 7. Lactose Fermentation Alkaline reaction Curd Formation (acid curd and rennin curd) Litmus reduction Proteolysis (Peptonization) Gas formation Acid Production

1. Lactose Fermentation: Organisms capable of using lactose as a carbon source for energy production utilize the inducible enzyme -galactosidase and degrade lactose as follows

The presence of lactic acid is easily detected because litmus is purple at a neutral pH and turns pink when the medium is acidified to an appropriate pH of 4. 2. Alkaline Reaction: An alkaline reaction is evident when the colour of the medium remains unchanged or changes to a deeper blue. This reaction is indicative of the partial degradation of casein into shorter polypeptide chains, with the simultaneous release of alkaline end products that are responsible for the observable color change.

3. Curd Formation: The biochemical activities of different microorganisms grown in litmus milk may result in the production of two distinct types of curds(Clots). Curds are designated as either acid or rennet, depending on the biochemical mechanisms responsible for their formation. Acid curd:-Lactic acid or other organic acid accumulation cause precipitation of the milk protein casein as calcium caesinate to form an insoluble clot. The clot is too hard and will not get retract from the walls of the test tube. An acid curd is easily identified if the tube is inverted and the clot remains immobile. Rennet curd:-Some organisms produce rennin, an enzyme that acts on casein to form paracasein, which in the presence of calcium ions is converted to calcium paracaseinate and forms an insoluble clot. Unlike the acid curd, this is a soft semi solid clot that will flow slowly when the tube is tilted. Acid or rennet curds are quite palatable dairy products known as Cottage Cheese. 4. Litmus Reduction: Fermentation is an anaerobic process involving biooxidations that occur in the absence of molecular oxygen. These oxidations may be visualized as the removal of hydrogen ( dehydrogenation) from a substrate. Since hydrogen ions cannot exist in the free state, there must be an immediate electron acceptor available to bind these hydrogen ions, or else oxidationreduction reactions are not possible and cells cannot manufacture energy. In the litmus milk test, litmus acts as such an acceptor. While in the oxidized state, the litmus is purple; when it accepts hydrogen from a substrate, it will become reduced and turn white or milk colored. This oxidation of lactose, which produces lactic acid, butyric acid, CO 2 and H2 is as follows:

The excess hydrogen is now accepted by the hydrogen acceptor litmus, which turns white and is said to be reduced. 5. Proteolysis (Peptonization): The inability of some microorganisms to obtain their energy by way of lactose fermentation uses other nutritional sources such as proteins for this purpose. By means of proteolytic enzymes, these organisms hydrolyze the milk proteins, primarily casein, into their basic building blocks, namely amino acids. This digestion of proteins is accompanied by the evolution of large quantities of ammonia, resulting in an alkaline pH in the medium with foul smell. The litmus turns deep purple. With further incubation of the medium, an opaque clearing of the medium occurs as the casein is hydrolyzed to peptides and amino acids. The opaque, turbid liquid supernatant (whey like appearance) turns brown in colour. 6. Gas Formation: Some bacteria(eg: Clostridia) ferment lactose of milk with the production of acid and gases like CO 2 and H2. Gas production is detected by bubbles in the acid curd or by the development of tracks or fissures within the curd as gas rises to the surface.

Litmus Reactions:

Procedure: 1. Using sterile technique, inoculate each experimental organism into its appropriately labeled tube by means of a loop inoculation. The last tube will serve as a control. 2. Incubate all cultures for 24 to 48 hours at 37C.

Expected Results:

Figure - Litmus milk reactions: (A) Alkaline. (B) Acid. (C) Upper transparent portion is peptonization; solid white portion in bottom is coagulation and litmus reduction; overall redness is interpreted as acid. (D) Coagulation and litmus reduction in lower half; some peptonization (transparency) and acid in top portion. (E) Litmus indicator is masked by production of soluble pigment.

Limitations: 1. Litmus milk is a complex medium that can produce a diversity of results. Because of this, litmus milk can give quite unreliable results (i.e., the results often give negative results when something positive should happen). Thus, you would be advised to use litmus milk as a confirmatory test but not a definitive test (except as a last resort). 2. A clot formation is simply recorded as clot and cannot clearly differentiate between a clot and curd formation in this medium.