Microbiology: Gram-Positive Bacilli Overview

Gram-Positive Bacilli

• Gram-positive bacilli are a diverse group of organisms found in the environment.
• They rarely cause human disease but are used in industry for enzymes and antibiotics.
• Some are important food spoilage organisms.

Non-Spore Forming, Non-Branching, Catalase Positive Bacilli

• The following organisms are non-acid fast.
• Most are part of the normal human flora.
• They colonize various parts of the human body.
• They are also found in the environment.
• Associated with various animals.
  • Corynebacteria
  • Rothia
  • Listeria

Corynebacteria General Characteristics

• Usually found in fresh and salt water, soil, and air.
• This genus includes many species of aerobic and facultative gram-positive rods.
• Closely related to Mycobacteria and Nocardia regarding cell wall composition.
• These organisms typically exhibit a meso-diaminopimelic acid (m-DAP) and mycolic acid cell wall.
• Lipophilic Corynebacteria are considered fastidious organisms.
• They may demonstrate slow growth on standard culture media.

Corynebacterium diphtheriae General Characteristics

• Most notable pathogen of Corynebacteria.
• Causative agent of diphtheria.
• Most common sites of infection: tonsils and pharynx.
• Virulence factor: Diphtheria toxin (DT).
• Produced by bacteria infected with a lysogenic B-phage, which carries the tox gene.
• Diphtheria toxin is heat-stable.
• Composed of two fragments: Fragment A (cytotoxicity) - active fragment. Fragment B- binding fragment.
• The toxin is not toxic until exposed to trypsin through trypsinization.
• This inhibits protein synthesis of cells.
• Enhanced considerably when grown in low iron conditions.

Clinical Infections

• To cause disease, Corynebacterium diphtheriae must invade and proliferate in local tissues.
• Transmitted by droplets, direct contact with cutaneous infections, and fomites.
• Characterized by a lesion ranging from a simple pustule to a chronic, non-healing ulcer.
• Respiratory diphtheria is pharyngitis with exudative membrane (pseudomembrane).
• Pseudomembrane is gray-white and adheres to the mucous membrane.
• Spread to nasal mucosa and pharyngeal wall causes neck edema.
• Respiratory diphtheria can cause "bull neck" appearance, which involves swelling of lymph nodes.

Treatment

• Antitoxin (commercially produced from horses) is the treatment to neutralize the toxin.
• Must be administered early, as it only neutralizes circulating toxin.
• Has no effect on toxin already fixed or within the cells.
• Corynebacterium diphtheriae is susceptible to antimicrobials like penicillin and erythromycin.
• Erythromycin is the most effective antimicrobial.

Loeffler's Methylene Blue Staining

• Demonstrates the presence of metachromatic granules (Babes-Ernst or Volutin granules).
• These granules take on bluish-purple in Loeffler's methylene blue stain.
• Often located at the poles of the bacilli (polar bodies).
• Composed of Polymetaphosphates and are more strongly gram-positive than other bacterial cells.

Biochemical Tests

• Catalase positive (+)
• Nitrate reduction (+)
• Urease test
• Beta hemolysis (variable)
• Gelatin hydrolysis (-)
• Esculin hydrolysis
• Carbohydrate fermentation(glucose (+), maltose (+), sucrose (-))

Cultural Characteristics

• Optimal growth at 37°C (15-40°C range), aerobe and facultative anaerobe.
• Corynebacterium species usually grow on 5% sheep's blood and chocolate agar.
• Media with enrichments (blood, serum, egg) necessary for good growth.
• Selective and differential media used if diphtheria is suspected.
• Usual media for diphtheria bacillus cultivation: Loeffler serum agar, CTBA, and Modified Tinsdale Agar.
• Loeffler's serum agar (or Pai agar) - enriched, rapidly growing colonies that can appear small at first and become large with a distinct yellow tint after incubation.
• CTBA (cystine-tellurite blood agar)- selective, differential, used to identify Corynebacterium diphtheriae.
• Corynebacteria form black or brownish colonies on CTBA due to reduction of tellurite to tellurium.
• Modified Tinsdale Agar - selective, differential.

Listeria General Characteristics

• Gram-positive psychrotrophic bacteria found in many environmental sources.
• Non-sporulating, catalase positive, indole and oxidase negative, and facultative anaerobic rods.
• Show motility at 25°C.
• Listeria monocytogenes is the most important species due to pathogenicity in humans and animals.
• Capable of surviving within phagocytes.

Listeria Pathogenesis

• Hemolysin (Listeriolysin O): major virulence factor, cytotoxic, hemolytic
• Internalin (Act A): cell-surface protein that induces phagocytosis.
• Phospholipase: L. monocytogenes ingested through contaminated food, producing Listeriolysin (O) and phospholipases.
• These allow the organism to escape from WBCs and spread to the bloodstream.
• Eventually reaches the central nervous system and placenta.

Listeria Clinical Infections

• Listeriosis - infections associated with Listeria monocytogenes.
• Common in pregnant women, newborns, and immunocompromised persons, usually during the 3rd trimester.
• Signs include spontaneous abortion, stillborn neonates, and newborn death.
• CNS infections (meningitis), endocarditis are also possible infections in immunocompromised hosts.
• Healthy individuals who ingest large amounts of Listeria monocytogenes can suffer from gastroenteritis.

Listeria Laboratory Diagnosis

• Microscopic: gram-positive rod or coccobacilli, singly or in chains, and tumbling motility.
• Observation of motility by direct wet mount to identify the organism.
• Culture: using blood, CSF, and swabs from lesions.
• Listeria from body tissues and fluids (e.g., blood, CSF) can grow.

Listeria Cultural Characteristics

• Optimal growth temp: 30-35°C.
• Commonly used media include: SBA, chocolate agar, NAP, and Brain-Heart Infusion Agar (BHIB).
• Beta-hemolytic, small, color white, smooth, and translucent colonies (1-2 days incubation).
• Cold enrichment (4°C several weeks).
• CAMP test (positive): "block" pattern.
• Rhodococcus equi, New Oxoid Chromogenic Listeria Agar: turquoise colonies with halos.

Listeria Differentiating Characteristics

• Pathogenic Listeria species: Listeria monocytogenes and Listeria ivanovii can be distinguished.
• Phospholipase: can hydrolyze lecithin from the culture medium, and an opaque zone or halo forms around colonies.
• Turquoise colonies with halos indicate Listeria monocytogenes.

Listeria Treatment and Prevention

• Penicillin with or without aminoglycosides.
• Resistant to tetracyclines.
• Prevention: avoid undercooked vegetables, meat, and soft cheeses and unpasteurized dairy products.
• Cook foods thoroughly and cook animal products thoroughly.

Erysipelothrix rhusiopathiae General Characteristics

• Domestic swines are the major reservoir.
• Found in animals (mammals, birds, fish).
• Human infections are rare; associated with fish handlers, farmers, and food preparation workers.
• Usual route: skin cuts and scratches.
• Infections are a result of a puncture wound or skin abrasion.

Erysipelothrix rhusiopathiae Clinical Infections

• Erysipeloid: localized skin infection, usually in hands or fingers, resembles streptococcal erysipelas, and is self-limiting (3-4 weeks, low incidence).
• Cutaneous infection (generalized, diffuse): rare, associated with endocarditis.
• Mortality rate is 38%.
• Immunosuppressed patients may develop bacteremia or septicemia.

Erysipelothrix rhusiopathiae Laboratory Diagnosis

• Specimens: tissue biopsy and aspirates of skin lesions.
• Microscopic: gram-positive, thin, pleomorphic rods (chains), singly (V shape).

Erysipelothrix rhusiopathiae Biochemical Tests

• Catalase negative, non-sporulating, non-motile.
• H2S positive, urease negative.
• Does not hydrolyze urea or esculin, Voges-Proskauer negative, but produce H2S and triple sugar iron agar.
• Differentiates from Lactobacillus, Listeria, and Kurthia
• Erysipelothrix does not grow at 4°C.
• Glucose and lactose fermenter.
• E. rhusiopathiae is negative. E. tonsillarum and E. inopinata are positive.

Erysipelothrix rhusiopathiae Culture

• Commonly plated on sheep blood agar or chocolate blood agar; patients with sepsis may be inoculated into broth with 1% glucose, incubated in 5% CO2 at 35oC.
• SBA or Chocolate agar: non-hemolytic or alpha-hemolytic, pinpoint colonies from systemic infections.

Erysipelothrix rhusiopathiae Treatment and Prevention

• Susceptible to sulfonamides, trimethoprim-sulfamethoxazole.
• Resistant to penicillin
• Treatment options include drainage and surgery.

Lactobacillus General Characteristics

• L. acidophilus, L. fermentum, L. vaginalis, L. salivarius, and L. plantarum.
• Normally isolated from dairy products.
• Also isolated from endocarditis cases.
• Reported cases of infections (endocarditis, bacteremia) associated with immunocompromised individuals (HIV) and gastrointestinal diseases.
• Important for maintaining proper pH balance in vaginal secretions.

Lactobacillus acidophilus General Characteristics

• Widely distributed.
• Normal flora in the human mouth, GI tract, and female genital tract (vaginal canal).
• Mostly encountered as a contaminant.
• Immunocompromised patients may develop bacteremia.
• Microscopically: highly pleomorphic bacilli, long chaining rods, and coccobacilli.

Lactobacillus Biochemical Tests

• Catalase -, oxidase -, indole -, nitrate -, hydrogen sulfide (-), and glucose, maltose, sucrose (+).
• Acidic pH of vaginal secretions inhibits growth of Mobiluncus, Prevotella, Gardnerella vaginalis, and Candida albicans.

Gardnerella vaginalis General Characteristics

• First described in 1953.
• Normal biota in the human urogenital tract.
• Naturally found in the vaginal area.
• Also colonizes the distal urethra of males.
• Anaerobic gram-positive rod.
• Gram-positive with a thinner peptidoglycan layer than typical Gram-positive bacteria.

Gardnerella vaginalis Clinical Infection

• Bacterial vaginosis (BV): characterized by an increase in vaginal pH (>4.5).
• BV is one of the most common causes of vaginal discharge.
• Associated with maternal and fetal morbidity during pregnancy.
• Characterized by foul smelling, excessive, and purulent vaginal discharge (malodorous discharge).

Gardnerella vaginalis Laboratory Diagnosis

• Specimen: vaginal discharge, isolated from urine (occasional cause of UTI).
• Microscopic: short, pleomorphic gram positive rod, staining gram-variable.
• Characteristic finding: presence of "clue cells."

Gardnerella vaginalis Cultural Characteristics

• Can Grow on 5% sheep blood and chocolate agar.
• Does not grow on MacConkey agar.

Gardnerella vaginalis Biochemical Tests

• Catalase -, oxidase -, hippurate hydrolysis +.
• Other tests include amine test or whiff test for odor, KOH solution as well as special selective, and differential media (HBT, cycloserine, cefoxitin, and fructose agar).
• Incubation is at 35 °C, in 5-10% carbon dioxide for 24-48 hrs

Nocardia General Characteristics

• Commonly found in soil.
• Implicated in human infections, usually in immunocompromised patients.
• Gram-positive, often with a beaded appearance, and partially acid-fast or modified acid-fast positive, catalase positive, and strictly aerobic organisms.
• Produces branching filaments.
• Mycolic acids are shorter chained in cell walls than mycobacteria.
• Partially acid-fast (stains with carbolfuchsin).

Nocardia Clinical Infections

• Pulmonary Nocardiosis: the most common clinical presentation, caused by inhalation, where it presents as chronic bronchopneumonia.
• Can also be acute or relapsing in immunocompromised patients.
• Cutaneous: Nocardia brasiliensis associated with primary cutaneous infections resulting from trauma.
• Skin infections, skin abscesses, lymphocutaneous infection or sometimes mycetoma.
• Mycetoma is a chronic, subcutaneous infection.

Nocardia Laboratory Diagnosis

• Specimen: sputum (thick, purulent, and no sulfur granules).
• Microscopy: branching filaments, fragmentation.
• Culture: may be pigmented, chalky, matte, or velvety; also found in dry, crumbly, and breadcrumb-like appearance.
• Nocardia species can grow on standard non-selective media.

Nocardia Treatment and Prevention

• Drainage and surgery in some cases.
• Antibiotics (sulfonamides, trimethoprim-sulfamethoxazole).
• Resistant to penicillin.

Spore-Forming Gram-Positive Bacilli

• Bacillus: obligate aerobes; spores are non-bulging.
• Clostridia: obligate anaerobes; spores are bulging.

Bacillus General Characteristics

• More than 100 species within the genus Bacillus.
• It is the largest gram-positive genus.
• Found widely in the environment, soil, important medically relevant organisms..
• Gram-positive, or gram-variable rods, in singles or pairs.
• Spores are not stained; often appear intracellular or extracellular clear oval structures like "empty spaces"
• Aerobic or facultative anaerobes
• Contains pathogens (Bacillus anthracis).
• peritrichous flagella; causing these organisms to be motile
• Except for Bacillus anthracis and Bacillus mycoides, which are non-motile organisms.
• Growth temperature range: -5°C to 75°C

Bacillus, Bacillus anthracis Clinical Characteristics

• Primary virulence factors are two exotoxins (e.g., edema factor, lethal factor).
• Edema factor causes edema/swelling, blocks phagocytosis.
• Lethal factor: kills macrophages, reducing immune defense of a host.
• Capsule is unusual (composed of amino acids rather than polysaccharide). Anti-phagocytic.
• Primarily a disease of herbivores.
• Endemic in developing countries in Africa, Central, and South America.
• Enzootic - the disease typically occurs among animals
• Spores are the source of infection spread (animal-to-animal; animal-to-human/environment); these come from contaminated soil, and contaminated animal products or animal carcasses.

Bacillus anthracis Clinical Infections

• Cutaneous anthrax: inoculation of endospores through skin breaks, resulting in a central black eschar (black necrotic lesion) surrounded by vesicles and an erythematous ring (malignant pustule)

• Pulmonary anthrax (inhalation anthrax):

• inhalation of spores.

• Respiratory infections resulting from exposure to endospores during handling of animal products.

• Flu-like symptoms to severe respiratory distress, dyspnea, cyanosis, pleural effusion, disorientation, and coma which can happen 24 hrs. after exposure.

• Gastrointestinal anthrax: ingestion of spores in contaminated meat.

• Results in abdominal pain, nausea, vomiting, and bloody diarrhea.

Bacillus anthracis Laboratory Diagnosis

• Specimen collection:
  • Blood, lung tissue (for pulmonary)
  • Fluids under eschar lesions (for cutaneous).
• Laboratory Microscopic and Cultural methods

Bacillus anthracis Treatment and Prevention

• Antibiotics to treat early-recognized anthrax promptly (e.g., penicillin, ciprofloxacin).
• Doxycycline can be used.
• General controls: deep burial or burning of animal carcasses; animal products are autoclaved; protective clothing/gloves to handle materials.

Bacillus cereus General Characteristics

• Other non-pathogenic Bacillus species
• Common soil and airborne contaminant
• Virulence: enterotoxins/spores
• Clinical infections: common cause of food poisoning (reheated rice or potatoes-diarrheal, emetic).

Bacillus cereus Types of Toxin

• Diarrheal toxin (heat-labile):

• Food contamination is usually from meat, poultry, vegetables, and dried beans.

• Incubation typically is 8-16 hrs.

• Emetic toxin (heat-stable):

• Food contamination is usually from fried rice.

• Incubation typically is 1-6 hrs.

Bacillus cereus Laboratory Diagnosis

• Microscopic: Gram-positive sporulating rod.
• Culture: SBA, beta-hemolytic, frosted glass appearance, large gray irregular colonies.
• Biochemistry test: Catalase positive, motility test (non-motile).
• Identify by hemolysis on BAP, lecithinase production, string of pearl test, growth on PEA, and/or gelatin hydrolysis.

Bacillus cereus Treatment and Prevention

• Food safety (preventing contamination of food items; proper food preparation/cooking times; and storage) avoidance of reheating.

Clostridium General Characteristics

• Saprophytes.
• Soil, freshwater, decaying matter, sewage, and other environmental sites.
• Gram-positive rods (but wider than vegetative bacteria), non-motile.
• Have bulging spores (subterminal position).

Clostridium botulinum General Characteristics

• Strictly anaerobic, gram-positive, motile, oval, and subterminal spores.
• Widely distributed saprophyte found in soils, vegetables, fruits, mud in lakes, and sea mud.
• Causative agent of botulism.
• Toxin production (e.g., Types A, B, E).
• Spores are heat-resistant.
• Improper food preservation/processing methods/heating can lead to toxin-generating organism growth.

Clostridium botulinum Clinical Infections

• Infant botulism (baby syndrome): 2-6 months.
• Ingesting spores in contaminated food (e.g., honey, food).
• Patients do not have the ability to suck and swallow.
• Characteristic signs include floppy neck, extreme weakness, and weakened voice.

Clostridium tetani General Characteristics

• Strictly anaerobic, gram-positive bacilli.
• Terminal, round spores.
• Appear as drumsticks.
• Widely distributed in soils.
• Causative agent for tetanus (acute disease).
• Clinical features:
• Skeletal muscle spasm, autonomic nervous system disturbance.
• Spores/vegetative bacteria produce tetanus toxin (tetanospasmin-neurotoxin).

Clostridium tetani Clinical Infections

• Tetanus (lockjaw, trismus): skeletal muscle spasm and autonomic nervous system disturbance due to germination of spores.
• Commonly is transmitted via contaminated breaks in skin by spores.

Clostridium perfringens General Characteristics

• Commensal in the large intestine.
• Gram-positive, non-motile, capsulated bacillus.
• Subterminal, bulging spores.
• Organism can multiply in tissue.
• Produces gas and multiple types of toxins.

Clostridium perfringens Toxins and Clinical Infections

• Food poisoning (most common), due to heat-stable enterotoxin.
• Incubations typically happen 6 to 15 hrs.
• Gastroenteritis (nausea, abdominal cramps, watery diarrhea).
• Gas gangrene (rapidly progressing, edematous myonecrosis; associated with contaminated deep wounds/trauma).

Clostridium perfringens Laboratory Diagnosis

• Microscopy: boxcar-shaped gram-positive bacilli.
• Culture on sheep blood agar with double zone hemolysis (inner zone of complete hemolysis surrounded by a much wider zone of incomplete hemolysis).
• The Direct Nagler Test to differentiate Clostridium perfringens.

Clostridium difficile General Characteristics

• Gram-positive spore-forming bacillus
• A major cause of hospital-acquired infections.
• Spores are found widely in nature, particularly in hospitals.
• Colonization in the colon of patients with prolonged hospital stays.
• Prolonged antimicrobial use disrupts the normal colonic flora.
• This increases susceptibility to C. difficile infection.

Clostridium difficile Clinical Infections

• Pseudomembranous colitis (colonic disease).
• Usually due to toxin-mediated pathogenesis.
• Associated with prolonged, and sometimes excessive use of antimicrobial therapies.

Clostridium difficile Laboratory Diagnosis

• Cultures on selective (different) media—e.g., CCFA or Cycloserine-Cefoxitin-Fructose Agar or egg yolk agar.
• Lecithinase (white zone of opacity), Lipase (iridescent), and Proteolytic E (complete narrow clearing).