Anaerobic Bacteriology: Clostridium & Gram-Negative

Introduction to Anaerobic Bacteriology

• Obligate anaerobes are microorganisms, that obtain energy exclusively through fermentation
• These organisms cannot utilize molecular oxygen for energy production
• Free oxygen can damage them, limiting their ability to colonize the human body and cause disease

Clostridium Genus

• Consists of gram-positive, spore-forming rods
• Associated with soft tissue, skin infections, antibiotic-associated colitis, and diarrhea
• Synthesizes potent exotoxins
• Specific species toxins cause botulism, tetanus, gas gangrene, and pseudomembranous colitis

Anaerobic Gram-Negative Bacteria

• Includes rods and cocci like Bacteroides, are frequently involved in visceral abscesses
• Often present in polymicrobic (mixed) infections with facultative bacteria
• Facultative bacteria utilize oxygen, which allows anaerobes to thrive

Clostridia Overview

• Anaerobic, gram-positive rods of significant clinical importance
• Clinically significant species include:
  • C. perfringens causes histotoxic infections (myonecrosis) and food poisoning
  • C. difficile causes pseudomembranous colitis associated with antibiotic use
  • C. tetani causes tetanus (lockjaw)
  • C. botulinum causes botulism

General Features of Clostridia

• Large, gram-positive, blunt-ended rods
• Form endospores, useful for species identification based on spore position within the vegetative cell
• Most species are motile
• Cannot use free oxygen as the terminal electron acceptor in energy production
• Use small organic molecules like pyruvate as final electron acceptors for energy

Clostridia and Oxygen Sensitivity

• Inhibited or damaged by O₂ in the vegetative state
• Produce oxygen radicals and peroxides, but lack enzymes like peroxidases, catalase, or superoxide dismutase to detoxify them
• Grow on enriched media with reducing agents like cysteine or thioglycolate to maintain low oxidation-reduction potential
• Can also grow in an O₂-free gaseous atmosphere (air-evacuated glove box, sealed jar)

Clostridia in the Environment and Infections

• Commonly found in the human environment, including potential pathogens
• Part of the intestinal flora in humans and other mammals
• Found in soil, sewage, and aquatic settings, especially those with high organic content
• Can cause destructive and invasive infections when introduced into tissues
• Infections are often opportunistic, deriving from the patient's own flora

Survival and Clinical Significance of Clostridia

• Endospore formation allows persistent survival due to resistance to chemical disinfectants
• Spores can withstand UV irradiation and boiling temperatures, but not standard autoclaving conditions (121°C for 15 minutes)
• Frequently occur as commensals or contaminants in clinical materials, complicating sample interpretations

Clostridium perfringens Characteristics

• Large, rod-shaped, non-motile, gram-positive, encapsulated bacillus
• Ubiquitous in nature, with its vegetative form in the normal vagina and gastrointestinal tract
• Spores found in soil (rarely seen in the body or after in vitro cultivation)
• When introduced into tissues, can cause anaerobic cellulitis and myonecrosis (gas gangrene)
• Some strains cause a common form of food poisoning

Pathogenesis of C. perfringens

• Secretes exotoxins, enterotoxins, and hydrolytic enzymes to facilitate disease
• Elaborates at least twelve exotoxins
  • Alpha toxin is crucial for virulence in tissues
  • Alpha toxin is a Lecithinase that degrades lecithin in mammalian cell membranes, causing lysis of endothelial cells, erythrocytes, leukocytes, and platelets

C. perfringens Exotoxins and Strains

• Other C. perfringens exotoxins induce hemolytic, cytolytic, and necrotic effects locally or when dispersed in the bloodstream
• Strains are grouped A through E based on exotoxins
• Type A strains produce both alpha toxin and enterotoxin, responsible for most human clostridial infections

C. perfringens Enterotoxins

• Enterotoxin is a small, heat-labile protein that acts in the lower portion of the small intestine
• The molecule binds to receptors on the epithelial cell surface, altering the membrane
• Disrupts ion transport (primarily in the ileum), leading to loss of fluids and intracellular proteins
• Enterotoxin-producing strains are usually heat-resistant; spores remain viable for over an hour at 100°C, enhancing their threat as enteropathogens

C. perfringens Degradative Enzymes

• C. perfringens is a metabolically vigorous organism that produces hydrolytic enzymes
• Enzymes include proteases, DNases, hyaluronidase, and collagenases
• Enzymes liquefy tissues and promote the spread of infection, enhancing pathogenesis

Clinical Significance of C. perfringens Infections

• Disease results from a combination of infection with exotoxins, enterotoxins, and degradative enzymes
  • Myonecrosis (gas gangrene)
    • Clostridial spores are introduced into tissues via dirt contamination or endogenous transfer from the intestinal tract

Factors in Myonecrosis (Gas Gangrene) Development

• Severe open wounds like compound fractures and necrotizing injuries from car accidents create disposing conditions
• Lowered tissue oxidation-reduction potential, resulting from cell injury or compromised circulation, allows spores to germinate and grow rapidly

Development And Effects of Gas Gangrene

• Lesions typically involve coinfection with clostridia, other anaerobes, and facultative species
• Facultative species consume available oxygen protecting anaerobes from oxygen's toxic effects
• Alpha toxin and other exotoxins are secreted, leading to extensive cell killing
• Enzyme production breaks down ground substances, facilitating the infection's spread

Progression of Gas Gangrene

• Fermentation of tissue carbohydrates yields gas, leading to gas bubbles in subcutaneous spaces and a crinkling sensation on palpation (crepitations), hence the name gas gangrene
• Exudates are copious and foul-smelling
• Increased capillary permeability allows exotoxins to circulate from damaged tissues to other organs
• Systemic effects include shock, renal failure, and intravascular hemolysis

Outcomes of Untreated Clostridial Myonecrosis

• Without treatment, gas gangrene is uniformly fatal within days of initiation
  • Anaerobic cellulitis
    • Clostridial infection of connective tissue
    • Bacterial growth spreads along the fascial plane (fasciitis)

Anaerobic Cellulitis Details

• Does not involve invasion of muscle substances
• Necrotic processes have a limited role
• Surgical intervention is typically unsuccessful because of the infection's rapid spread

C. perfringens Food Poisoning

• A common cause of food poisoning
• Symptoms: nausea, abdominal cramps, and diarrhea, typically start eight to eighteen hours after consuming contaminated food
• Fever is absent and vomiting is rare
• The attack is usually self-limited, with recovery within one or two days

Sources And Conditions For Food Poisoning

• Common vehicles for clostridial food poisoning are meat, chicken, fish, and their byproducts
• Clinical symptoms require a large inoculum of 10⁸ or more organisms
• Atypical episodes involve cooking that fails to inactivate spores, followed by holding the food for several hours under conditions that allow bacterial germination

Other C. perfringens Related Diseases

• Enteritis necroticans
  • A necrotizing bowel disease with high mortality caused by C. perfringens
  • Known as pigbel in New Guinea and dambrand in Germany
  • Clinically similar illnesses are rare in the United States

Laboratory Identification of Clostridial Infections

• Diagnosis of clostridial myonecrosis or cellulitis relies heavily on clinical impression
• The presence of clostridia in clinical materials may be due to accidental contamination
• Specimens from diseased tissue, when Gram-stained, typically show vegetative clostridial forms accompanied by other bacteria and cellular debris

C. perfringens Culture and Identification

• When cultured anaerobically on blood agar, C. perfringens rapidly grows colonies, with a unique double zone of hemolysis
• In food poisoning cases, the organism can be sought in suspected food and patient feces
• Quantitative cultures showing heavy growth (greater than 10^5 organisms/gram of food or feces) suggest causal significance

Treatment and Prevention of C. perfringens

• Key to prevention and treatment of gas gangrene: immediate removal of foreign materials, devitalized tissues, and exposure to oxygen
• Amputation may be necessary if debridement cannot control gangrene progression
• Supplement with high doses of antibiotics, as C. perfringens is sensitive to Ampicillin
• Broad-spectrum antibiotics are essential due to potential mixed infections

C. botulinum and Botulism

• Causes botulism, with several clinical forms
• Botulism is due to a potent neurotoxin
• Causes flaccid paralysis
• Direct contact with the organism is unnecessary; the disease can be caused by the toxin alone

Epidemiology of C. Botulinum

• Widely found in soil and aquatic sediments
• Spores frequently contaminate vegetables
• Outbreaks often occur in families or eating groups due to toxin elaboration in food
• Organism germinates under alkaline conditions

Pathogenesis of Botulism

• Multiple botulinum toxins are designated A through G
• Human diseases are mainly caused by types A, B, and E
• Botulinum serotypes and tetanus toxin are homologous proteins with neurotoxicity arising from proteolytic cleavage of specific synaptic vesicle peptides, leading to neurotransmission failure

Mechanism of Botulinum Toxin

• Contrast to tetanus toxin, which causes contraction (spasms), botulinum toxins block peripheral cholinergic synapses at neuromuscular junctions
• Inhibits the release of the neurotransmitter, acetylcholine, preventing contraction and causing flaccid paralysis
• Minute doses of botulinum toxin preparations are used therapeutically to relieve spastic conditions like post-spasticity and strabismus

Clinical Significance of Botulism

• Classic botulism
• Results from food poisoning
• Initial Symptoms: difficulties focusing vision, swallowing, & impaired cranial nerve functions.
• Occurs 12-36 hours after ingesting toxin contaminated food

Progression of Botulism

• No fever or signs of sepsis are present
• Progressive paralysis of striated muscle groups develops, with a mortality rate of about 15%
• Respiratory paralysis is the usual cause of death
• Recovery is typically protracted, taking several weeks

Infant Botulism

• The most common form of botulism in the United States, causing floppy baby syndrome
• C. botulism colonizes the large bowel of infants (3-24 weeks old), toxin is slowly absorbed
• Common early signs: constipation, feeding problems, lethargy, or poor muscle tone

Risks for Infant Botulism

• Certain formula supplements, like honey contaminated with C. botulinum spores, may transmit the organism
• Possibly a cause of sudden infant death syndrome (SIDS)
• Wound botulism: A rare form of botulism that occurs when a wound is contaminated with the organism, and the toxin is absorbed from the site

Botulism Treatment and Prevention

• Administer antitoxin, to neutralize unbound botulinum toxin, as soon as possible if botulism is suspected
• A trivalent (A B E) horse antiserum is available
• Supportive measures, such as mechanical ventilation, may be required

Clostridium tetani Introduction

• Introduction of C. tetani into a dirty wound is a common occurrence
• Due to extreme O₂ sensitivity of vegetative C. tetani and widespread immunization against its exotoxin, the resulting disease, tetanus, is rare
• Typically seen in older individuals and some infants whose immunity from vaccination has waned

Tetanus Development and Sources

• Growth of the organism is completely local
• Produces a powerful neurotoxin that causes spastic paralysis after being transported to the central nervous system
• C. tetani spores are frequently found in soils and gardens
• The most typical site for infection in tetanus is a punctured wound caused by a splinter
• Tetanus spores germinate and multiply in devitalized tissues

Tetanus Infection Scenarios

• Special circumstances may cause infections
• Examples: severe burns or surgery
• Illicit drugs
• Spores are introduced by injection
• In developing countries
• Tetanus is a common complication of birth
• Umbilical wounds are treated with animal dung or unsterilized equipment

Pathogenesis of Tetanus

• Tetanus toxin, called tetanospasmin
• Potent toxin
• Transported to the central nervous system
• Retrograde neuronal flow or by blood
• Plasmid-coded exotoxin
• Single antigenic type
• The mature toxin (formed two chains/disulfide bond)

Tetanus Toxin Action

• Heavy Fragment (B)
  • Mediates binding to neurons
  • Cellular penetration of Light Fragment (A)
• A Fragment
  • Blocks neurotransmitter release by inhibiting synapses prolonged muscle spasm
  • Protease
  • Cleaves synaptic vesicle protein, stops signal flow

Clinical Significance of Tetanus

• Incubation Period
  • 4 days - several weeks
• Shorter Period
  • More severe disease
  • Wound/brain proximity
• Spastic Paralysis
  • Site of infection

Symptoms and Progression of Tetanus

• Early Stages : lock jaw/trismus occurs because of jaw muscle affectation
• Voluntary Muscles: Gradually become involved.
• Any Stimulus
  • Noise/Bright Light
  • Painful Spasm & Convulsions

Mortality related to Tetanus

• Death rate
  • 15 - 60%
  • Paralysis of Chest muscle
  • Respiratory Failure

Laboratory identification of Tetanus

• Diagnosis is largely based on clinical findings
• The focus of infection is often a trivial wound
• C. tetani unique
  • Slender rod shape
  • Round terminal Spore morphology
  • Swarming on anaerobic blood agar

Treatment for Tetanus

• Prompt antitoxin administration to neutralize free Toxin
• Prefer treatment with human hyperimmune globulin (tetanus immune globulin)
• Vigorous care with sedatives, muscle relaxants, and attention to ventilation.

Eradicating and preventing Tetanus

• The organism is sensitive to penicillin, and this drug can be used to eradicate the infection.
• Prophylaxis is necessary to minimize infection of tetanus wounds
• Vaccination is important: active immunization with tetanus toxoid and DPT with antigens for diphtheria toxoid and pertussis

Clostridium difficile Introduction

• Diarrhea is a common complication of antimicrobial & antineoplastic drug treatment.
• Conditions go from loose stools to life-threatening pseudomembranous colitis (PMC).
• C. difficile accounts for ¼ of antibiotic-associated diarrheas (AAD) in hospitalized patients and almost cases of PMC.

C. difficile Colonization and Risk

• Two-three % of healthy adults have the bacteria as part of their natural flora
• High presence of this bacteria amongst nursing home patients
• Spores of C. difficile spread in dust and on surfaces
• New residents/immunocompromised patients at risk
• Higher risk for intestinal side effects due to antibiotics.

C. difficile pathogenesis

• C. Difficile minor component of gut flora
• Antibiotics suppress healthy species
• Pathogenic bacteria release two toxic polypeptides, A & B:

C. difficile Toxin A & B

• Toxin A enterotoxin/fluid excretion & stimulates inflammatory response.
• Cytopathic effects with tissue culture. Toxin B disrupt protein synthesis + cause disorganization of the cytoskeleton
• Toxic in tissue culture.

C. difficile: Clinical Significance

• Antibiotics cause clostridial AAD and colitis.
• Clindamycin, ampicillin, and cephalosporins.
• Symptoms include:
• Mild diarrhea
• Inflammation
• Fulminant PMC.

C. difficile Symptoms & Progression

• The pseudomembranous exudate is composed of mucus, fibrin inflammatory cells debris + ulcer epithelium. -Best located using an endoscopy
• mild symptoms can last after treatment
• Infants in hospitals quickly colonized but rarely have diarrhea.

Laboratory Identifying C. difficile

• C. difficile identified cultured anaerobically
• Toxin stool extracts show toxin production
• Recently developed ELISA for toxins A/B immunoassays

Treatment for C. difficile

• If symptoms are mild
• Stop antibiotics
• Stay hydrated
• Recurring problems warrant antibiotics
• Metronidazole
• Vancomycin

Anaerobic Gram Negative Rods

• Common organism in the oral cavity ( gingiva), female genital tract + lower GIT
• Present in Infections
• Ten % bacteremia

Anaerobic Gram Negative Rod infections

• Majority bacteria w/ anaerobic abscesses.
• Can breach body tissue w/infection establishment
  • Ruptured appendix/trauma
• Compromised immune status

Types of abscesses

• Localized abscess (common) w/ species of organsims.
• Facultative bacteria lower oxygen + create anaerobic
• Bacteria include gram-negative rods

Bacteroides species

• Most common anaerobe in the human colon.
• Part of normal flora
• Caused by blood during bowel penetration/trauma-surgery.
• Commonly caused by anaerobic organisms

Bacteria makeup/transmission

• Coccobacilli bacteria
• Polysaccharides vital for resistance
• Travels colon-blood (abdominal trauma).
• infection=endogenous (not person to person)

What is B. fragilis?

• Primary cause bacteria for infections as well - B. fragilis -Lipopolysaccharides- heparinase destroy tissue

B. fragilis Bacteremia

• Quickly multiples + leads to bacteremia.
• With abdominal cavity intro.
• Peritonitis + abdominal abscess

Laboratory ID with gram -anaerobes

• Infections foul smelling
• Specimen collection important
• Oxygen sensitive + transported anaerobically -Some bacteriodes = O2 tolerant

How to ID gram - bacteria

• Slender rods with mixed flora
• Hard with debris polymorphonuclear
  • Culture blood/anaerobic blood agar

Treatment & prevention w/gram - bacteria?

• resistance = common w/bacteria
• Metronidazole = best -Ampicillin
• Aminoglycosides ineffective + surgical drainage w/abscess

Prevotella

• Mouth / Alamentary
• Respiratory tract /Endotoxin Common infections with sinus and dental. Also upper respitory such as abscesses/and Plumonary

Prevotella Infection

• Source human bite- oral Flora
• Black colonies on blood agar
• Prevotella has lower resistance

Fusobacteria

• Pointed end spindle Shape
• Normal: mouth/colon/GIT
• Slow grower low virulent Great in great variety for peritonitis- abdominal Also empyema, osteomyelitis etc