COURSE MLSM 505 LECTURE TOPIC: GRAM POSITIVE BACILLI GENUS: BACILLUS Dr. John Ochei

1. COURSE MLSM 505 LECTURE TOPIC: GRAM POSITIVE BACILLI GENUS: BACILLUSDr. John Ochei

2. Lecture Objectives • At the end of the lecture, the student shall be able: 1. Name the diferrent species of the genus Bacillus 2. Name four infections caused by the bacteria 3. Perform laboratory diagnosis of anthrax 4. Determine the antibiotic susceptibility test on the organisms.

3. General Characteristics: This group of organisms consists of large aerobic spore-bearing rods. They are Gram positive bacilli which may sometimes be arranged in long chains. Most of the species are motile.

4. Type species: B. anthracis (anthrax bacillus). • It is the main pathogen of the group. • Other important species that may cause infections are B. cereus and B. subtilis. • Several other species are mainly saprophytes. Some of them are strict aerobes.

5. B. anthracisBacillusanthracis is primarily a pathogen of herbivores. The organisms are excreted in the faeces, urine and saliva of infected animals. They form spores which can remain viable in the environment for years. B. anthracis produces 2 types of toxins. A. a toxin complex made up of three factors-oedema factor, a 'protective antigen' and lethal factor. These are plasmid encoded and they act synergistically. B. An anti-phagocytic capsular polypeptide which is also plasmid encoded.

6. Saprophytic species: The other species of the genus Bacillus, including B. cereus and B. subtilis are saprophytes found in the soil, water, vegetation and foodstuffs such as meat, milk, cereals and spices. They form spores which are found almost everywhere including hospitals, laboratories and food factories.

7. Some of these spores e.g. those of B. subtilis, are very resistant to disinfectants, stains and boiling at 1000C for several hours but are killed by autoclaving at 1210C for 15 minutes. • B. subtilis is one of the commonest aerobic spore bearers (ASBs).

8. Pathogenicity B. anthracis causes 1. Cutaneous anthrax: This begins as a single painless blister which is usually referred to as 'malignant pustule'. Infection is generally by spores entering through damaged skin. The spores germinate in the tissue at the site of entry and spread via the lymphatics to blood stream causing septicaemia, toxaemia and death. .

9. Herdsmen, butchers and those involved in treating animal skins are most at

10. 2. Pulmonary anthrax: • This is caused by inhalation of the organism or spores. It causes a severe haemorrhagic condition in the bronchi and lungs which may result in septicaemia and death. The condition is referred to as "wool sorters disease" as those engaged in wool and hides treatment are mostly infected

11. 3. Enteric anthrax: This is caused by ingestion of contaminated foodstuffs such as meat and milk. It is a severe form of gastro-enteritis which progresses to septicaemia, toxaemia and death. It is not a common condition. Meningitis is a complication of anthrax septicaemia.

12. B. cereus It causes: 1. Food poisoning which may result from eating of either contaminated cooked rice or other cereals, or meat. The symptom is onset of vomiting and diarrhoea within a few hours or up to 24 hours depending on the kind of contaminated food eaten. It produces 2 types of enterotoxins - a diarrhoeal enterotoxin and an emetic toxin; both are associated with food poisoning. B. cereus also produces a lecithinase ( acts on the phospholipids on the cell membrane ), and haemolysins ( cereolysin and cytolysin ). 2. Pneumonia and wound infections. These conditions are not common.

13. B. subtilisThis species has been reported to cause meningitis, endocarditis and other infections in debilitated and immunosuppressed patients.

14. Animal infection and inoculation with B. anthracis: Herbivores and other animals are generally infected by ingesting contaminated grass and other type of vegetation. The spores, on ingestion, germinate in the tissue and get into the blood stream. A full post mortem on a dead animal is not encouraged. The carcass is disposed of by burning according to the guidelines of the Public Health Authority.

15. Identification of B. anthracis can be confirmed by inoculating the thigh of a guinea pig with fluid from a suspected malignant pustule or 0.5 ml broth culture of the organism. The animal dies with 48-72 hours after inoculation. Post-mortem shows gelatinous exudate at the site of inoculation; and enlarged, congested dark red spleen. Smears of blood and spleen material show characteristic appearance and positive Mcfadyean's reaction.

16. Laboratory diagnosisSpecimens Specimens include pus or fluid from the pustule, sputum, blood, spinal fluid and swabs from cutaneous lesions.Caution: All procedures should be carried out in the safety cabinet: B. anthracis is a highly infectious organism and must be handled with care. The specimens must carry the biohazard labels.

17. MicroscopyGram stained smears from the specimens show large Gram positive rods of about 5-8 x 1.5 um with square or round ends and are without spores. Blood film stained with polychrome methylene blue shows capsulated large bacilli mostly in chains. The capsule material stains purple. This is McFadyean's reaction. B. anthracis capsule is a polypeptide unlike most other capsules which are polysaccharides.

18. Smears from cultures show large bacilli containing spores which are of the same diameter as the bacilli. The spores are centrally placed. The smears are chemically fixed and not by heat, by immersing the dry smears in 4% potassium permanganate for about 10 -15 minutes. This is to ensure that the organisms arecompletely dead.Motility test: B. anthracis is non-motile while the other species are generally motile.

19. CultureThe Bacillus species grow well on most culture media, aerobically at 35-370C within a range of 12-450C. On blood agar after 24 hours incubation, B. anthracis produces large (2-3 mm in diameter), grey, opaque irregular colonies with wavy edges (Medusa head). Colonies are non- haemolytic or slightly haemolytic. When grown in the presence of a high concentration of CO2, it produces smooth mucoid colonies.Broth cultures of B. anthracis contain thick pellicles on the surface of the medium, with no turbidity. Other species produce turbidity in broth culture.

20. B. cereusA selective medium is used to isolate it from faeces. Mannitol egg yolk phenol-red polymyxin agar is recommended. Colonies after 24 hours incubation are large( 3-5 mm), flat, dry and grey surrounded by white precipitates. B. cereus is beta haemolytic on blood agar.

21. Identification testsB. anthracis ferments glucose, maltose, sucrose and trehalose with acid and no gas production. It is catalase positive, nitrate positive and unlike other species, liquefies gelatine slowly in an "inverted fir tree" pattern. Other distinguishing features of the Anthrax bacillus from other species are as follows:

22. B. anthracisOther species Non-motile Generally motile Capsulated Non capsulated No turbidity in broth Often turbidity in broth Slow liquefaction of gelatine Rapid liquefaction of gelatine Inverted fir tree form No inverted fir tree form Some strains of Bacillus produce little lecithinase.

23. Antibiotic sensitivity: The organisms are sensitive to penicillin, tetracycline, chloramphenicol and cotrimoxazole. Some useful saprophytic species 1. B. licheniformis -used for the production of penincillinase. B. polymyxa -used for the production of antibiotic, 2. polymyxin B. 3. B. stearothermophilus -used for testing the efficiency of autoclaves.