Microbiology

Mary Ann C. Bunyi, MD

Genus Bacillus Large gram positive rods occurring in chains Most are saprophytic organisms (such as Bacillus subtilis and Bacillus cereus ) occurring in soil, water, air and on vegetation Bacillus anthracis is the principal pathogen of the genus and causes anthrax About 50 species Soil organisms Three (3) important Bacillus sp. o Bacillus subtilis o Bacillus cereus o Bacillus anthracis Two (2) species of major medical importance o Bacillus cereus o Bacillus anthracis

Lecture 14 (2-3): Bacillus August 24, 2011

EPIDEMIOLOGY Ubiquitous in the environment Found in many foods, especially rice, pulses (legumes), meat and vegetables Present in small numbers in raw, dried, and processed foods Spores are heat resistant therefore survive pasteurization, brief cooking or boiling Vegetative forms produce enterotoxins TRANSMISSION Ingestion of food/drinks contaminated with organism or toxin DISEASES Food poisoning from reheated cooked rice and meat/vegetables Gastroenteritis Two (2) clinical syndromes associated with Bacillus cereus food borne illness due to two (2) different toxins 1. Emetic syndrome o Acquired by eating food with preformed toxin most commonly fried rice or recooked rice o Occurs after a short incubation period (0.5 6 hours) o Nausea, vomiting, abdominal cramps o Diarrhea seen in one third of patients o Caused by heat-stable toxin 2. Diarrhea syndrome o Spore-associated disease caused by ingestion of contaminated meat or vegetables o Slightly longer incubation period (6 24 hours) o Predominantly characterized by moderate to severe abdominal cramps and watery diarrhea o Vomiting seen in one fourth of patients o Caused by heat-labile enterotoxin Both syndromes are mild, not associated with fever and abate within 24 hours Table 1. Summary of two (2) clinical syndromes associated with Bacillus cereus food borne illness
Emetic Syndrome Diarrhea Syndrome

** Bacillus subtilis was not discussed due to medical insignificance; however, it is included in this transcription for additional information. MORPHOLOGY Typical cells measure 1 x 3-4 m Have square ends Spores are in the center Aerobic, gram positive rods, occurring in chains See Figure 4, Color Plates, page 4 PHYSICAL PROPERTIES Saprophytic species use simple sources of nitrogen and carbon for energy and growth Spores are resistant to environmental changes such as dry heat and chemical disinfectants Animal products contaminated with anthrax spores can be sterilized only by autoclaving Bacillus subtilis Hay bacillus or grass bacillus Gram positive Catalase positive Model organism for laboratory studies Not considered a human pathogen; though it may contaminate food but rarely causes food poisoning Spores can survive the extreme heating that is often used to cook food, and it is responsible for causing ropiness a sticky, stringy consistency caused by bacterial production of long-chain polysaccharides

CHARACTERISTICS Large, gram-positive, spore-forming rod Motile Non-encapsulated Respires aerobically Bacillus cereus CHARACTERISTICS Large, gram-positive, spore-forming rod Motile Non-encapsulated Respires aerobically Self-limited LABORATORY IDENTIFICATION Non-fastidious Hemolysis on horse and sheep blood agar double zone of beta hemolysis (See Figure 5, Color Plates, page 4) Cream to white colonies (See Figure 6, Color Plates, page 4) Lecithinaes positive Mannitol negative Policarpio. Rabino. Sagun. Sistoso. Sobremisana. Sorbito.

Source Incubation period Manifestation Duration Toxin Responsible

Rice < 6 hours Severe vomiting 8-10 hours Heat-stable toxin

Meat/Vegetables > 6 hours Diarrhea 20-36 hours Heat-labile enterotoxin

OTHER MANIFESTATIONS Local skin and wound infections, periodontitis, ocular infections and invasive disease including bacteremia, catheter-associated infections, endocarditis, osteomyelitis, pneumonia, brain abscesses, and meningitis Ocular involvement such as panophthalmitis, endophthalmitis and keratitis INVASIVE RISK FACTORS Intravenous drug use, indwelling catheters or implanted devices, neutropenia or immunosuppression, or preterm birth Bacillus cereus endophthalmitis occurs after penetrating ocular trauma and intravenous drug use PATHOGENESIS Heat-stable toxin o Associated with spore germination o Present in food contaminated with Bacillus cereus o Vomiting within 1-5 hours after ingestion Heat-labile enterotoxin MICROBIOLOGY Lecture 14 (2-3)| Page 1 of 4

o Acquired after ingestion o Produced by Bacillus cereus in the intestine o Diarrhea within 10-15 hours after ingestion DIAGNOSIS Isolation of the organism in a concentration of > 105 colony forming unit (CFU) per gram of incriminated food Organism in feces or vomitus of ill people is not a definitive evidence of infection unless isolates from several ill patients are demonstrated to be the same serotype or unless stool cultures from a matched control group test negative Toxin testing is not widely available Phage typing, DNA hybridization, plasmid analysis, enzyme electrophoresis are used as epidemiologic tools in foodborne outbreaks Those with risk factors, isolation of the organism from wounds, blood or other sterile body fluids is significant TREATMENT For foodborne illness, supportive treatment only since illness is short-lived and self-limiting Removal of infected foreign bodies For invasive disease, give antibiotics o Drug of choice is vancomycin o Alternative drugs include clindamycin, carbapenems, ciprofloxacin PREVENTION Hygienic preparation of food Cooked food should be stored in the refrigerator and reheated thoroughly before serving Bacillus anthracis From the Greek word anthrakis which means coal and referring to the most common form of the disease, cutaneous anthrax, in which large black skin lesions are formed Major disease threat to herbivores (cattle, sheep, horses, hogs, goats) Humans are infected by: 1. Direct contact with diseased animal, hides, wool, brushes, or bone meal 2. Inhalation of infected dust (Woolsorter's disease) 3. Ingestion of infected meat

Spores formed in culture, soil, and dead animal tissues but not in live animals Spores remain viable in soil for decades Encapsulates in 5% CO2 atmosphere Only bacterium with a protein capsule (D-glutamate)

CULTURE CHARACTERISTICS Round, cut glass appearance Hemolysis is uncommon (while among saprophytic bacilli, hemolysis is common) See Figure 9, Color Plates, page 4 GROWTH CHARACTERISTICS Animal products as hides, bristles, hair, wool, bone contaminated with anthrax spores can be sterilized only by autoclaving EPIDEMIOLOGY Incidence o A zoonotic disease that occurs in rural regions in the world Transmission o Spores remain viable in soil for decades Source of infection for livestock through ingestion o Human infection is through contact with infected animal or animal products o Outbreaks of gastrointestinal anthrax is associated with ingestion of undercooked or raw meat o Can be used as a biological weapon because 1. Spores are highly stable 2. Spores can infect via the respiratory route resulting in inhalational disease that is lethal 3. Spores can also be introduced into food products or water supplies INCUBATION PERIOD For all forms of anthrax, less than 2 weeks In inhalational anthrax, because of spore dormancy, there is slow clearance from the lungs and incubation period may be prolonged to as long as several months FORMS OF ANTHRAX 1. Cutaneous 2. Pulmonary 3. Gastrointestinal 1. Cutaneous anthrax Accounts for more than 95% of human cases Spores enter the broken skin, germinate and rapidly proliferate at the site of entry Vesicular papule at the site of infection Blue-black edema Rupture of papule will reveal a black eschar (malignant pustule) See Figure 10, Color Plates, page 4 2. Inhalational Anthrax Spore inhalation and phagocytosis Naturally occurring inhalational anthrax was historically a disease associated with wool sorters at industrial mills A result of the deposition of spore-bearing particles of 1 to 5 m into the alveolar spaces Disease immediately follows germination Germination, replication, and infection of hilar lymph nodes Chest X-ray shows lobulated mediastinal widening, which may have infiltrates (See Figure 11, Color Plates, page 4) Abrupt onset of respiratory failure (1-2 days later) Hemorrhage, edema, and necrosis are the results of bacterial toxins released during replication. The first stage of illness is flu-like with fever, myalgia, dyspnea, cough, headache, vomiting, chills, abdominal pain, and chest pain. Sudden fever, dyspnea, diaphoresis, and shock characterize the second stage of illness. In the second stage of illness, cyanosis and hypotension result in a rapid death Death usually results 2-3 days after the onset of symptoms Chest radiographs showing a widened mediastinum are evidence of hemorrhagic mediastinitis and pleural effusions MICROBIOLOGY Lecture 14 (2-3)| Page 2 of 4

Figure 1. How Bacillus anthracis affects humans HISTORICAL BACKGROUND The first disease to fulfill Kochs postulate in 1876 The first bacterial disease for which immunization was available (1881) Review Kochs postulates RECENT CASES OF ANTHRAX IN THE U.S (2001) Two postings of contaminated letters (4 were found) 22 cases (plus 1 laboratory acquired case) 11 inhalational and 11 skin cases 5 deaths 32,000 were given prophylactic antibiotics Extensive contamination of postal system Thousands of false alarms and hoaxes MORPHOLOGY & PHYSIOLOGY Gram-positive, aerobic, non-motile, spore-forming bacilli with square ends tending to form long chains Bamboo canelike (See Figures 7 and 8, Color Plates, page 4) Policarpio. Rabino. Sagun. Sistoso. Sobremisana. Sorbito.

3. Gastrointestinal Anthrax Not seen in the United States Ingestion of infected meat Proliferation within the gastrointestinal tract Fever, acute abdomen, vomiting, bloody diarrhea Presence of intestinal eschar (similar to cutaneous lesion) Spreads to mesenteric lymph nodes, septicemia, shock and death PATHOGENESIS (VIRULENCE FACTORS) Capsule o Coded as pX02 plasmid o Polypeptide of D-glutamic acid o Encoded by a plasmid o Antiphagocytic o Not a good immunogen o Antibodies to the capsule are not protective against the disease Exotoxins o Coded as pX01 plasmid o A plasmid-encoded, heat-labile, heterogeneous protein complex made up of 3 components 1. Edema factor (EF) 2. Lethal factor (LF) 3. Protective antigen (PA) o In vivo, these three factors act synergistically (for toxic effects). The protective antigen binds to surface receptors on eukaryotic cells and is subsequently cleaved by a cellular protease. The larger C-terminal piece of PA remains bound to the receptor and then binds either EF or LF, which enters the cell by endocytosis. Edema Factor, when inside the cells binds calmodulindependent and acts as adenylate cyclase.

o o o o

Large gram-positive rods Nonmotile Nonhemolytic Rapid screening assay (PCR and antigen-detection based) for use on cultures and directly on clinical specimens Confirmatory criteria for identification of Bacillus anthracis Capsule production and visualization and lysis by gamma-phage Direct fluorescent antibody assays (DFA) for capsule antigen and cell wall-associated polysaccharide

TREATMENT Prompt therapy Protect against the multiplication of the organism Protect against the effects of the anthrax toxin, if there is septicemia Ciprofloxacin o The antibiotic of choice for victims of terrorism or warfare o 400 mg IV q12h Doxycycline o 100 mg IV q 12h Pen G o Endemic anthrax o 2M U IV q2h o 4M U IV q4h Postexposure prophylaxis o Ciprofloxacin 500mg po q 12h o Doxycycline 100 mg po q12h o Vaccine x 3 doses at 0, 2 and 4 weeks o Starting antibiotics within 24 hours after aerosol exposure is expected to provide significant protection Duration of 60 days with or without vaccine Most effective when combined with vaccination Antibiotics are still indicated even when fully immunized Long-term antibiotics necessary because of spore persistence in lung/lymph node tissue PREVENTION Antibody to the toxin complex is neutralizing and protective Two vaccines available for humans (1) and animals (1) o Immunize cattle and other herbivores o Immunize humans at risk SUMMARY Presumptive indentification
Clinical specimen (blood, CSF, etc.)

Figure 2. Pathogenesis of Edema Factor o Lethal factor's mechanism of action involves activation of macrophages and production of cytokines which cause necrosis, fever, shock and death. Individually, the three proteins have no known toxic activity. Antibodies to protective antigens prevent PA binding to cells stop EF and LF entry.

Gram stain Figures 7 and 8

Isolate on SBA Figure 12

Figure 3. Pathogenesis of Lethal Factor DIAGNOSIS Presumptive o History and clinical findings o Gram positive bacilli on blood smears Definitive o Culture and identification o After incubation on a blood agar plate for 1224 hours at 3537oC, well-isolated colonies are 25 mm in diameter and heavily inoculated areas may show growth in 68 hours o Gray-white, flat or slightly convex colonies are irregularly round, with edges that slightly undulate, and have ground glass appearance o Often have comma-shaped protrusions from colony edge Medusa head colonies (See Figure 9, Color Plates, page 4) Laboratory Criteria for Identification o From clinical samples, such as blood, cerebrospinal fluid (CSF), skin lesion (eschar), or oropharyngeal ulcer, microscopy and gram stain will show encapsulated gram-positive rods o From growth on sheep blood agar Policarpio. Rabino. Sagun. Sistoso. Sobremisana. Sorbito.
Colony morphology Hemolysis, Motility, Spores Figure 13 Gram stain

Confirmatory identification
Isolate

Phage lysis Figure 14

Capsule

DFA

Horse blood (MFadyean Stain) Figure 15

Bicarbonate Media (MFadyean stain India ink stain) Figure 16

Capsule antigen Cell wall

Figure 17

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COLOR PLATES

Figure 10. Cutaneous anthrax. Rupture will reveal a black eschar (malignant pustule).

Figure 4. Gram stain of Bacillus sp. Large, gram-positive bacilli with squared-off ends occurring singly and in chains. Some species demonstrate endospores, which may be located centrally or terminally along the bacilli.

Figure 11. Mediastinal Widening and Pleural Effusion on Chest X-Ray in Inhalational Anthrax

Figure 5. Bacillus cereus with double zone of beta hemolysis

Figure 12. Isolate on 5% sheep blood agar

Figure 13. Motility of Bacillus anthracis Figure 6. Colonies of Bacillus cereus on 5% sheep blood agar. Cream to white colonies and beta hemolysis.

Figure 14. Phage lysis

Figure 7. Gram stain of Bacillus anthracis

Figure 15. Bacillus anthracis with MFadyean Stain

Figure 8. Gram-positive Bacillus anthracis in the CSF

Figure 16. Bacillus anthracis on Bicarbonate Media

Figure 17. Capsule antigen cell wall of Bacillus anthracis Figure 9. Medusa head colonies of Bacillus anthracis Policarpio. Rabino. Sagun. Sistoso. Sobremisana. Sorbito. MICROBIOLOGY Lecture 14 (2-3)| Page 4 of 4

Policarpio. Rabino. Sagun. Sistoso. Sobremisana. Sorbito.

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