Anaerobes 2006 07 Med

Anaerobic Bacteria

Objectives • Differentiate obligate anaerobes from facultative organisms • Describe how anaerobes as part of normal flora, initiate and establish infection. • To know the clinical infections associated with the following organisms and how they are acquired and manifest  Clostridium spp.  Bacteriodes spp  Fusobacterium spp  Actionmyces  Gram-positive cocci • To know the laboratory methods of performing cultures and identifying anaerobes • Acceptable and unacceptable specimens • Antimicrobial therapy

Anaerobic Bacteria • Why anaerobic bacteria are important?? • Because they play a role in serious, often fatal, infection and intoxications

Anaerobic isolates based on Gram stain morphology Gram stain Ve+

Bacilli

Cocci .Peptococcus spp .Peptostreptococcus spp

Spore No yes Clostridium spp

Ve -

Cocci

Bacilli

.Bacteriods spp .Fusobacterium spp Veillonella spp .Porphyromonas spp .Prevotella spp .Actinomyces spp .Bifdobacterium spp .Eubacterium spp .Lactobacillus spp .Propionibacterium spp

Relationship to Oxygen and carbon Dioxide Obligate aerobes 15-20% oxygen

Microaerophile 5% oxygen

Facultative anaerobe grows equally well in the presence or absence of oxgyen

Aerotolerant anaerobe grows in reduced concentration of oxygen

Strict anaerobe 0% oxygen, most bacteriods many clostridium

ANAEROBIC BACTERIA A. SPORE-FORMING Genus Clostridium • C. perfringens • C. tetani • C. botulinum • C. difficile

B. NON-SPORING • Bacteroides • Fusobacteria • Actinomyces

Are usually present as normal flora of colon of animals

Are an important part of normal flora of mouth, colon, skin & lower genital tract

CLOSTRIDIUM PERFRINGENS • Gram-positive • Spore-forming bacilli with square ends • Seven (7) types (A-G) on the basis of exotoxins • Type A is the most important in humans • Spores are located in soil • Vegetative cells are normal flora of colon & vagina

CLOSTRIDIUM PERFRINGENS CULTURAL CHARACTERS • Anaerobic • Grow on blood agar producing two zones of hemolysis ° Complete hemolysis due to O toxin ° Incomplete hemolysis due to alpha-toxin

• Ferments sugars with production of acids & gas ° Much gas is also produced in vivo in necrotic tissues hence the disease is called “Gas Gangrene”

CLOSTRIDIUM PERFRINGENS ENZYMES • Collagenase • DNAase • Protease • Hyaluronidase

TOXINS

Responsible for invasiveness

1. Alpha-toxin (Lecithinase) • Hydrolyses lecithin & sphingomyelin • Disrupts cell membranes of RBCs, WBCs & muscle cells

CLOSTRIDIUM PERFRINGENS TOXINS 2. O Toxin o Causes Lysis of RBCs o Is toxic for muscles 3. Enterotoxin • Causes food poisoning

DISEASES By C. PERFRINGENS 1. Gas Gangrene (Myonecrosis)

Occurs in traumatic open wounds contaminated with soil Open wounds contaminated with soil

Pathogenesis of Gas Gangrene Large amount of gas in subcutaneous tissues

Spores germinate Organisms multiply

Alpha Toxin cell death & tissue necrosis

Reduced Oxygen due to tissue necrosis O Toxin Cause muscle death

Enzymes Break down sugars with gas

DISEASES By C. PERFRINGENS 1. Gas Gangrene (Myonecrosis) Prevention & Treatment • Surgical toilet and excision of necrotic tissues • High doses of penicillin • Place patient in hyperbaric oxygen chamber to: ° Inhibit bacterial growth ° Neutralize toxicity of O toxin

• Amputation of limb if necessary to prevent spread of disease

DISEASES By C. PERFRINGENS 2. Food Poisoning • Spores from soil contaminate food (especially meat dishes) • Survive cooking, germinate & multiply in gut • Produce enterotoxin - food poisoning • Incubation Period : 8-24 hours • Diarrhea & abdominal cramps

LAB DIAGNOSIS

• Mainly Clinical • Gram-stain of specimen • Culture

CLOSTRIDIUM TETANI • Gram-positive bacilli • Spore-forming with terminal spores (drum-stick appearance) • Spores remain viable in soil for years ° Destroyed by autoclaving at 121oC for 15 min

Exotoxin produced • A neurotoxin (tetanospasmin) • Causes tetanus

TETANUS Mode of Transmission • Contamination of wounds with soil • Road-side accidents • Nail penetration of foot Can be contamination of : • Umblicus & circumcision wounds - tetanus neonatorum • Surgical wounds - Post-surgical tetanus

Pathogenesis of Tetanus Occurs in traumatic open wounds contaminated with soil Wounds contaminated with soil Spores germinate in dead tissues Neurotoxin Reaches retrograde to spinal cord

Muscle spasm • Organisms multiply locally only • No tissue damage • No invasion of adjacent tissues

Continuous excitement of motor neurons Inhibits release of inhibitory mediators

Binds to ganglioside receptors of anterior horn cells

TETANUS : Clinical features Incubation Period

• Few days to weeks, depends on: ° Site of wound ° Condition of wound

Clinical Picture

• Lock jaw (inability to open mouth) • Intermittent spasm of muscles of respiration & swallowing • Generalized convulsions & opisthotonus • Death from exhaustion & respiratory failure

TETANUS : Treatment o Care of wound o Tetanus immunoglobulins to neutralize unbound toxin Intramuscular & intrathecal o Penicillin G to prevent further multiplication of bacilli

Supportive measures:

• Care of respiration • Sedation with benzodiazepines to prevent spasm

TETANUS : Prevention ACTIVE IMMUNIZATION OF: 1. Infants as part of triple vaccine (DPT) 2. Pregnant women to protect newborn 3. All road side accident wounded patients

CLOSTRIDIUM BOTULINUM Morphology • Gram-positive, motile bacilli • Spore-forming with oval subterminal spores Habitat • Spores are found in soil and sediments of lakes world-wide

CLOSTRIDIUM BOTULINUM Exotoxin Production

• One of the most potent exotoxins known • Lethal dose may be just 1µg • Heat-labile - destroyed at 100oC easily

DISEASE BY C. BOTULINUM : BOTULISM

Source of Infection • Consumption of contaminated homecanned food (Do not grow in acidic canned foods) • The organisms multiply during storage • Produce exotoxin without change of color, odor or taste of food

Pathogenesis of botulism Occurs due to consumption of contaminated home-canned food Ingestion of contaminated home-canned Food

Flaccid paralysis

No excitement of motor neurons

Pre-formed exotoxin in food

Exotoxin absorbed from gut

& Enter blood reach peripheral nerve synapses

Inhibits release of acetylcholine

BOTULISM : Clinical Features Incubation Period : 18-96 hours • Vomiting • Paralysis of muscles including ocular, pharyngeal, laryngeal & respiratory • High death rate due to paralysis of respiratory muscles

INFANT BOTULISM • Organisms introduced with dietary supplement • Organisms multiply in colon with absorption of small amount of toxin • Infant suffers from constipation & feeding problems • One of the causes of sudden infant death

BOTULISM : Treatment & Prevention • Large doses of antitoxin • Supportive measures • Adequate sterilization of food during canning • Adequate heating before consumption

CLOSTRIDIUM DIFFICILE • Normal flora of adult colon (2-4%) • Also present in environment TOXINS PRODUCED Toxin A (Enterotoxin) • Causes mucosal damage with diarrhea • Helps absorption of toxin B Toxin B (Cytotoxin) • Inhibits protein synthesis and leading to cell death & pseudomembrane formation. The two toxins appear to act synergistically by unknown mechanism

Clostridium difficile-associated Diarrhea Predisposing Factors • The risk is highest with clindamycin, the cephalosporins, and ampicillin • It is lowest with vancomycin, metronidazole, and the aminoglycosides • Increased risk with the use of proton pump inhibitors • Cancer chemotherapy even in the absence of antibiotic therapy.

CLOSTRIDIUM DIFFICILE DISEASE: Antibiotic-Associated Pseudomembranous Colitis • Use of broad-spectrum antibiotics like clindamycin alter the normal flora of colon • Clostridium difficile will flourish • Causes Pseudomembrane (yellow-white) formation with poor absorption and diarrhea-like symptoms

CLOSTRIDIUM DIFFICILE • • • • •

DIAGNOSIS

Endoscopy to see pseudomembrane Bacterial culture Detection of toxin B in stool by cell cultures ELISA to detect toxins A & B Detection of glutamate dehydrogenase, a metabolic enzyme expressed at high levels by all strains of C. difficile, both toxigenic and nontoxigenic.

TREATMENT

• Antibiotic withdrawal • Oral vancomycin or metronidazole

Clostridium difficile

GRAM-NEGATIVE ANAEROBES BACTEROIDES • • • •

Gram-negative small bacilli Grow on selective media anaerobically Normal flora of colon & oral cavity Present in enormous numbers in faeces (1010/gm) • Common in cervix & vaginal fornices

GRAM-NEGATIVE ANAEROBES BACTEROIDES : Diseases Abscess formation o After abdominal & gynecological surgery o Brain abscess o Dental caries Treatment o Metronidazole OR Cl o Cefoxitin, cefotetan, and ceftizoxime have an enhanced antianaerobic spectrum. o Imipenem-cilastatin, meropenem, and erzapenem are the most broadly active

GRAM-NEGATIVE ANAEROBES • • • • •

FUSOBACTERIA

Gram-negative, spindle-shaped bacilli Normal flora of colon, oral cavity & female genital tract

Diseases

Chronic ulcerative lesions of gums Vincent,angina

Treatment

Metronidazole

PREVOTELLA/PORPHYROMONAS • • •

Previously in genus Bacteroides Found in oral cavity & colon Cause periodontal infections

Fusobacterium sp

Bacteroides fragilis

LAB IDENTIFICATION OF ANAEROBES • Transport • Without delay • Use anaerobic transport media

Anaerobic incubation Anaerobic jars • The most frequently used system • Uses hydrogen and CO2 generator envelope Anaerobic chambers • A final gas mixture of 5% CO2, 10% hydrogen and 85% nitrogen is used

LAB IDENTIFICATION OF CLOSTRIDIA Specimens • Pus & infected tissue • Blood, vomit & left-over food for detection of toxin in botulism • Feces & food for culture in C. perfringens food poisoning • Feces for C. difficile Gram-staining • Gram-positive, sporing bacilli Culture anaerobically on: • Anaerobic blood agar (Brucella BA) • Cooked meat medium Gas liquid Chromatography (GLC) Identifying anaerobes through metabolic end product analysis

Anaerobic, Gram-positive cocci Peptostreptococcus sp.

Propionibacterium acnes

.

Bifidobacterium bifidum

Actinomyces sp

LAB IDENTIFICATION OF CLOSTRIDIA Biochemical Reactions by:

Culture on Egg-Yolk Agar (EYA) is used to determine the activities of the following enzymes  Lecithinase  Lipase  Proteolyticinase activity

Bactroides • Specimens o Pus (foul smelling), blood

• Gram-staining o Gram-negative bacilli

• Culture o Bacteriod Bile Eusculin (BBE) o Kanamycin-vancomycin-lacked blood (KVLB) o Anaerobic incubation for 48-96 hours

Clinical learning assessment A 45 years male farmer was admitted to the hospital for complications resulting from a tractor injury to his left leg. The patient’s leg was painful, bluish, and edematous.. A radiograph revealed pockets of gas in the tissue. A complete blood count revealed a marked increase in neutrophils and a total WBC count of 33,000/mm3 . A Gram stain of the wound specimen revealed numerous rectangularly-shaped Gram –positive bacilli, moderate Gram –positive cocci in cluster, no spores, and very few leukocytes. The attending physician concluded that the Gram stain results were indicative of contamination rather than infection. Fortunately for the patient, the infectious disease specialist thought differently

Questions • What disease did the infectious disease specialist suspect (give two names for the disease) • What genus of bacterium do you suspect is causing the disease? • What species do suspect? • What is the clinical significance of the Grampositive cocci? • If this an infectious disease, why were so few leukocytes observed on the Gram stain?

Case Ms. A., an 18 years-old student, was admitted to hospital with diffuse abdominal pain, diarrhea, and nausea without vomiting. Her pain was localized to the right side of the abdomen. Physical examination reveals tenderness in the right lower quadrant of her abdomen. She received a first-generation cephalosporin antibiotic (cefazolin) and was taken to the operating room, where her ruptured appendix was removed. Cultures of the peritoneal in the area of the appendix grew a mixture of bacteria , typical of those found in stool. On the second after the operation, her temperature spiked to 38.5 0C. Blood cultures obtained preoperatively grew E. coli. Ms. A., improved postoperatively and complained a 7-day course of cephalosporin. Because she had no further symptoms and her blood cultures were negative, the antibiotic was stopped. However, 36 hours later her temperature was 38.5 0C and she felt diffuse pain over the site of appendectomy. A CAT scan of her abdomen revealed a retroperitoneal abscess. Cultures obtained after drainage of the abscess grew B. fragilis. She was again treated with antibiotics. This time metronidazole was used for 14 days, and Ms. A had recovered

The case raises several questions •

How did the two episodes of Ms. A’s disease with regards to pathogenesis and the kind of bacteria involved?

•

Why did B. fragilis survive the first course of antibiotic treatment?

•

Was Ms. A treated properly? What could have been done to lessen the likelihood of abscess formation?

•

How does B. fragilis facilitate intra-abdominal abscess formation?