Bacillus

Gram positive, motile, spores found in soil, water, air and common contaminants of bacteriological media.Two important pathogenic species are B.anthracis and B.cereus. Genus Bacillus has numerous applications such as:

·         Filter paper strips impregnated with spores of B. subtilis have been used to test the efficacy of hot air oven, and B. staerothermophilus to test the autoclave and low-temperature steam-formaldehyde sterilizer. B. globigi used to test EtO sterilizer, B. pumilis to test the efficacy of ionising radiation.

·         These produce antibiotics such as bacitracin, polymyxin and gramicidin and act as biological controls in assays of folic acid, aflatoxins and hexachlorophane.

·         B. thuringiensis has  been widely used as an insecticide mainly for pest  control  on food crops.

·         B. subtilis and other species have provided a model for microbial genetics.

The resistance of spores of Bacillus species to drying, UV, heat and chemical disinfectants make these organisms troublesome contaminants of food leading to a variety of food spoilage defect and pharmaceutical products. Their absence from these products acts as a good indicator of cleanliness of the product.

B.anthracis: Largest of pathogenic bacteria (4-8 X 1-1.5µm), found singly,pairs or chains,the entire chain being surrounded by a capsule which is polypeptide in nature. It is not formed under ordinary conditions of culture but only on media containing serum or bicarbonate in the presence of excess CO₂. When blood films containing anthrax bacilli  are stained with polychrome methylene blue  for a few seconds and examined under microscope, an amorphous purplish material is noticed around the bacilli. This represents the capsular material and is characteristic of anthrax bacilli. This is known as McFadyean's reaction and is employed for the presumptive diagnosis of anthrax. When stained with Giemsa's stain, the bacillus stains purple and capsule red.

                    Spores are formed in the culture or in the soil, but never in the animal body during life. Sporulation can be encouraged by distilled water and 2% NaCl and inhibited by anaerobic conditions and CaCl₂. The spores are refractile, oval, central in position and of the same diameter of the bacilli so that they do not cause bulging of the vegetative cell. In cultures, the bacilli are arranged end to end in long chains, with ends truncated, so that a chain of bacilli represents a “bamboo-stick” appearance.

                    It is an aerobe, facultative anaerobe, temp range 12-45°C. Nitrates reduced catalase produced. Optimum pH for growth is 7-7.4. They can grow on ordinary media. On Nutrient agar  colonies are round, raised, dull, opaque, grayish white, 2-3mm diameter with an irregular, fringe-like edge. Colonies on blood agar produce very slight hemolysis. Under low power, the edge of the colony is found to be composed of long, interlacing chains of bacilli, resembling curled hair-lock. This gives them the “medusa-head” appearance.

                    A selecive medium consisting of heart infusion agar with polymyxin, lysozyme, EDTA & thallous acetate-PLET medium devised to isolate the ant bacilli from other spore producers. In a gelatin stab culture there is growth down the stab line with lateral spikes that are longest near the surface, giving inverted fir tree appearance.

                    Vegetative  bacilli  destroyed  at  60°C  in  30  minutes. In  the  carcasses of animals  which have died of anthrax, the bacilli remain viable in the bone marrow for a week and in the skin for 2 weeks. Normal  heat  fixation  of  smears  may not  kill  the  bacilli  in  blood films. The spores are  highly  resistant  to physical and chemical agents. The anthrax bacillus is susceptible to sulfonamides,penicillin,erythromycin,chloramphenicol.

Antigenic Structure -Three main antigens have been charactrized:

1.      Capsular Polypeptide- It is polypeptide consisting exclusively of D-glutamic acid. Antibody to this are not protective.

2.      Somatic Polysaccharide- it is a component of the cell wall. It is made up of  NAG and D- galactose. It cross reacts with blood group A antigen. The antibody against this antigen is also non protective.

3.      Anthrax Toxin- It is a complex exotoxin consisting of 3 protein components: Protective antigen(PA), Lethal factor(LF), Edema Factor(EF). PA is capable of inducing protective antibody, it binds to surface receptors on sensitive eukaryotic cells thereby allowing translocation of EF and LF into the cell. EF is an adenyl cyclase, capable of generating high levels of cAMP in the cytoplasm of susceptible cells leading to edema.

Pathogenicity-Anthrax is a zoonosis that affects mostly plant-eating animals such as sheep, goat, cattle. Meat eating animals infected by eating infected flesh or inhaling anthrax spores. The endospore forms only under aerobic conditions and are not found in tissues or circulating blood. Most human anthrax results from contact with endospores during occupational exposure on farms,industries that handle wool, hides, meat, bones.This type is known as Respiratory Anthrax or Wool-Sorter’s Disease.

Human anthrax are of three types:

1.      Cutaneous

2.      Pulmonary (Respiratory)

3.      Intestinal

Cutaneous anthrax: About 95% of human cases of anthrax are cutaneous infections,by entry through skin (face, neck, hands, arms). The lesion starts as a papule 1-3 days after infection and becomes vesicular, containing fluid. The whole area is congested and swelled, lesions filled with serum (yellow fluid) are seen around a central necrotic lesion which is covered by a black eschar. The lesion is called a malignant pustule. The disease is common in dock worker’s carrying loads of hides and skins on their bare backs and known as Hide- Porter’s Disease. Spontaneously it heals,while some develop fatal septicemia.

Pulmonary anthrax: Wool-Sorter’s disease,common in worker’s in wool factories, due to inhalation of dust from infected wool. This is fatal, regardless of treatment. Clots form inside pulmonary capillaries and lymph nodes, cause swelling and that obstruct airways.

Intestinal anthrax: Rare, resembles symptoms as in food poisoning.A violent bloody diarrhea with high case of mortality.

In all the three cases there may be invasion of blood stream and localisation in the meninges,resulting in fatal meningitis. Anthrax infection in human beings provides immunity, secondary attacks are extremely rare.

Laboratory Diagnosis- Anthrax is diagnosed by culturing blood or examining smears from cutaneous lesions, sputum, gastric aspirates, faeces of patients. All procedures connected with the handling should be carried out with greatest care in a safety cabinet. Laboratory personnel should wear protective gowns, masks and surgical gloves when processing the samples. After the work, all laboratory work benches must be sterilized with 5% hypochlorite or phenol and all instruments should be autoclaved. Smears can be prepared and stained with Gram's  and McFadyean's methods and observed. Specimens can be cultured on nutrient agar, blood agar, PLET medium and nutrient broth. Serological diagnosis by ELISA may be of value.

                    The disease is treated with Penicillin. Patients hypersensitive to penicillin may be given ciprofloxacin, tetracyclin, chloramphenicol, erythromycin and gentamicin. A vaccine is available and given to workers annually, but industries should maintain dust free environments and provide respirators to prevent endospore inhalation. Clothing worn by workers should be sterilized and cleaned at the facility to prevent family members from becoming infected by handling it.

                    Animal immunization is an important means of prevention. Farmers must avoid using bone meal contaminated with anthrax spores, and must dispose by burying infected animals in deep, lime-limed pits. Lime prevents earthworms  from bringing anthrax endospores to the surface. Incineration is used, but must be done properly to avoid the spread of bits of contaminated carcass and spores.

B.cereus:  Causative agent of food poisoning. It is widely distributed in nature, isolated from soil, vegetables and a wide variety of foods such as milk, cereals, spices, meat and poultry. It is motile, non-capsulated.

B.cereus produces  two patterns of food-borne diseases.

1.      Cooked meat and vegetables, characterized by diarrhea and abdominal pain ie,8-16 hrs after ingestion.

2.      Consumption of cooked rice, characterized by acute nausea and vomiting 1-5 hrs after meal.

Two types of toxins: Emetic toxin- a heat stable toxin, patient develops nausea and vomiting with in 1-16 hours of ingestion. Enterotoxin- a heat labile toxin, acts on the epithelial cells of small intestine resulting in stimulation of cell bound adenyl cyclase, leads to increased levels of cAMP resulting in active secretion of large volumes of fluid into the intestinal lumen. This causes abdominal pain and profuse diarrhea.

                    Diagnosis of food poisoning is usually based on finding 10⁵ or more organisms per gram of incriminated food.