04 - ANAEROBES, MISCELLANEOUS BACTERIOLOGY & VIROLOGY (MLS Exam #4) Flashcards
Define
Anaerobe
A bacterium able to replicate in the absence of oxygen.
Define
Aerotolerant anaerobe
Anaerobes that can survive a short exposure to oxygen but will not be able to perform metabolic processes unless placed into an anaerobic environment.
Define
Obligate or strict anaerobe
Anaerobes that are killed almost immediately in the presence of oxygen.
Define
Exogenous anaerobes
Anaerobes that exist outside the bodies of animals and the infections they cause are termed exogenous infections.
They can be found in soil, in freshwater and saltwater sediments,
Exogenous anaerobic infections are usually caused by gram-positive, spore-forming bacilli belonging to the genus Clostridium. Clostridia initiate infection when spores are ingested by way of contaminated food or gain access to the body through open wounds contaminated with soil.
Define
Endogenous anaerobes
Anaerobes that exist inside the bodies of animals and are the source of endogenous infections.
The anaerobes most frequently isolated from infectious processes in humans are those of endogenous origin.
Discuss the significance of anaerobes in the infectious process.
- Anaerobes outnumber aerobes on mucosal surfaces, such as the linings of the oral cavity, gastrointestinal (GI) tract, and genitourinary (GU) tract. These heavily colonized surfaces are the usual portals of entry into the tissues and bloodstream for endogenous anaerobes.
- Under ordinary circumstances, microorganisms that are members of the microbiota do not cause disease, and many actually can be beneficial.
- However, when some of these organisms gain access to usually sterile body sites, such as the bloodstream, brain, and lungs, they can cause serious or even fatal infections.
Define normal anaerobic flora for specimen types
Skin
Indigenous members of the skin microbiota include anaerobes that colonize the sebaceous glands and hair follicles.
Propionibacterium, peptostreptococci, Cutibacterium (Propionibacterium) acnes
Define normal anaerobic flora for specimen types
Intestinal tract
- It is estimated that 500 to 1000 different species of bacteria live in the GI tract. Microbiota studies have found that anaerobes outnumber facultative anaerobes by a factor of 1000 : 1.
- Any infection in the peritoneal cavity would likely be caused by organisms that have escaped from the GI tract.
Bifidobacterium, Eubacterium/ Eggerthella, Clostridium, peptostreptococci, Bacteroides fragilis group, Parabacteroides, Bilophila, Campylobacter, Fusobacterium, Porphyromonas, Prevotella, Sutterella, Veillonella
Define normal anaerobic flora for specimen types
Upper respiratory tract and Oral Cavity
Of the bacteria present in saliva, nasal washings, and gingival and tooth scrapings, 90% are anaerobes.
Actinomyces, Eubacterium/ Eggerthella, peptostreptococci, Campylobacter, Fusobacterium, Prevotella, Bifidobacterium, Porphyromonas, Veillonella
Define normal anaerobic flora for specimen types
Genitourinary tract
- Although anaerobic bacteria colonize the distal urethra, they are not considered to be a cause of uncomplicated urinary tract infections.
- Similarly, 50% of the bacteria in cervical and vaginal secretions are anaerobes.
- GU swabs and voided or catheterized urine specimens are unacceptable for anaerobic bacteriology because recovery of these organisms would not distinguish whether they were present as pathogens or as endogenous microbiota.
Peptostreptococci, Bifidobacterium, Fusobacterium, Lactobacillus, Mobiluncus, Prevotella, Veillonella
Discuss
Factors That Predispose Patients to Anaerobic Infections
Generally, infectious diseases involving anaerobic bacteria follow some type of trauma to protective barriers such as the skin and mucous membranes. Trauma at these sites allows anaerobes of the endogenous biota (or in some cases, soil anaerobes) to gain access to deeper tissues.
Human or animal bite wounds. Aspiration of oral contents into the lungs after vomiting. Tooth extraction, oral surgery, or traumatic puncture of the oral cavity. Gastrointestinal tract surgery or traumatic puncture of the bowel. Genital tract surgery or traumatic puncture of the genital tract. Introduction of soil into a wound.
Discuss the physical characteristics that are found when anaerobes are recovered in the infectious process.
*Infectious processes involving anaerobes are usually purulent, with many polymorphonuclear leukocytes present. (not always)
* Infection in close proximity to a mucosal surface
* Presence of foul odor
* Presence of large quantity of gas
* Presence of black color or brick red fluorescence
* Presence of sulfur granules
Many of the infectious processes involving anaerobes consist of mixtures of obligate anaerobes or mixtures of obligate or aerotolerant anaerobes and facultative organisms, this is called what?
Polymicrobial infection
Associate the causative anaerobic organism with the clinical infection.
Food poisoning
Clostridium perfringens
C. perfringens is associated with two types of food poisoning— type A, a relatively mild and self-limited GI illness, and type C, a more serious but rarely seen disease. C. perfringens foodborne disease usually follows the ingestion of large numbers of enterotoxin-producing strains in contaminated food.
Associate the causative anaerobic organism with the clinical infection.
Botulism
Clostridium botulinum
- Results from the ingestion of preformed botulinum toxin,Botulinum toxin is an extremely potent neurotoxin; only a small amount produces death.
- Weakness and paralysis are the main features of botulism.
- The food sources involved commonly in botulism include home-canned goods.
- Botulinum toxin type A (Botox) is also used medically to treat chronic migraines and temporarily improve facial wrinkles.
What is a common source of infant botulism?
Raw Honey
Associate the causative anaerobic organism with the clinical infection.
Tetanus
Clostridium tetani
- Tetanus occurs when spores in the environment enter the skin through puncture wounds.
- The clinical manifestations of tetanus are attributed to the neurotoxin tetanospasmin produced by Clostridium tetani. Tetanospasmin acts on neurons, preventing the release of inhibitory and excitatory neurotransmitters. This results in a spastic type of paralysis, with continuous muscular spasms. leading to trismus (lockjaw), risus sardonicus (distorted grin), and difficulty breathing.
- Therapy for tetanus requires the injection of antitoxin, muscle relaxants, and intensive therapy.
Associate the causative anaerobic organism with the clinical infection.
Myonecrosis (gas gangrene)
C. perfringens (the most common cause)
- Myonecrosis, or gas gangrene, usually occurs when organisms contaminate wounds, through trauma or surgery.
- C. perfringens, C. histolyticum, C. septicum, C. novyi, and C. bifermentans have all been associated with myonecrosis.
- C. perfringens is the most common cause. Under favorable conditions, the organisms are able to grow, multiply, and release potent exotoxins. In gas gangrene exotoxins, such as α-toxin produced by C. perfringens, cause necrosis of the tissue and allow deeper penetration by the organisms.
- The onset and spread of myonecrosis can be rapid, and extensive surgical debridement of the necrotic tissue is often required.** If treatment is delayed, amputation of the affected limb is not uncommon.**
Associate the causative anaerobic organism with the clinical infection.
Pseudomembraneous colitis
Clostridioides difficile
- This organism is part of the GI biota in about 5% of individuals
- Following antimicrobial therapy, many bowel biota organisms other than C. difficile are killed, thus allowing C. difficile to multiply with less competition and produce high levels of two toxins: toxin A, an enterotoxin, and toxin B, a cytotoxin.
- Bloody diarrhea with associated necrosis of colonic mucosa is seen in patients with pseudomembranous colitis. C. difficile is a common cause of health care– associated (nosocomial) infection.
Associate the causative anaerobic organism with the clinical infection.
Actinomycosis
Actinomyces spp. (A. israelii is the most common)
Actinomycosis is a chronic, granulomatous, infectious disease characterized by the development of sinus tracts and fistulae, which erupt to the surface and drain pus that may contain so-called sulfur granules, dense clumps of bacteria that may be colored.
Associate the causative anaerobic organism with the clinical infection.
Lemierre’s disease
Fusobacterium necrophorum
Lemierre disease, a syndrome of thrombophlebitis of the jugular vein that occurs rarely following group A streptococcal pharyngitis, is caused by F. necrophorum. The streptococcal infection produces a peritonsillar abscess containing a number of bacterial species. The abscess aids F. necrophorum in penetrating the tissue to reach the jugular veins.
Associate the causative anaerobic organism with the clinical infection.
Acne
Propionibacterium acnes
Which nonselective media should be used for anaerobic cultures?
Anaerobic blood agar (CDC)
An enriched medium containing sheep blood for enrichment and detection of hemolysis, vitamin K (required by some Porphyromonas spp.), and yeast extract
What selective media is used to select for Bacteroides fragilis group?
Bacteroides bile esculin agar (BBE)
- Containing gentamicin (which inhibits most aerobic organisms)
- 20% bile (which inhibits most anaerobes)
- Esculin; used primarily for rapid isolation and presumptive identification of members of the B. fragilis group
- Black colonies (because of esculin hydrolysis)
Name the selective medium containing sheep red blood cells and phenylethyl alcohol; used primarily to suppress the growth of any facultative, gram-negative bacilli (e.g., Enterobacteriaceae) that might be present in the clinical specimen, especially swarming Proteus spp.
Phenylethyl agar (PEA)
Supports growth of almost all obligate anaerobes (gram-positive and gram-negative)
What is the composition of anaerobic gas?
- 5% hydrogen
- 80-90% nitrogen
- 5-10% carbon dioxide (CO2)
Name 3 ways in which ANO2 conditions can be acheived?
- Anaerobic chambers
- Anaerobic jars
- Anaerobic bags
How long should primary anaerobic cultures incubate before an initial read?
48 hours
How long are anaerobic cultures routinely held for?
5-7 days (5 days at CCF)
* 10-14 to rule out Actinomyces or Cutibacterium acnes
When anaerobes are suspected, what steps must be performed and recorded for each colony morphotype present on the anaerobic blood agar plate to initiate presumptive identification of the isolates?
- Describe the colony morphology and note the media in which growth occurred. (CDC, BBE)
- Describe the Gram stain reaction and cell morphology.
- Set up an aerotolerance test
- Perform MALDI-TOF (if available)
Define
Aerotolerance Testing
Determines whether a microorganism isolated under anaerobic conditions is a strict or facultative anaerobe.
Subculture the suspected isolate to both aerobic (BAP) and anaerobic (CDC) media and observe the actual atmospheric requirements of the organism.
Interpretation of Aerotolerance Test Results
Describe the principle and application of the test.
Fluorescence
Porphyromonas and Prevotella fluoresce brick red under long-wave (366-nm) UV light. (Wood’s lamp)
Describe the principle and application of the test.
Catalase
Helps differentiates aerotolerant strains of Clostridium (catalase negative) from Bacillus (catalase positive).
15% hydrogen peroxide
Describe the principle and application of the test.
Spot indole test
The spot indole test is useful in identifying C. acnes (indole positive) from the similar Propionibacterium spp. (indole negative).
Describe the principle and application of the test.
Urease test
C. sordellii is the only Clostridium species that is urease positive.
Describe the principle and application of the test.
Lipase production (EYA)
**Lipase-positive organisms produce a colony covered with an iridescent, multicolored sheen, sometimes described as resembling the appearance of gasoline on water or mother of pearl.
Fusobacterium necrophorum
Describe the principle and application of the test.
Lecithinase production (EYA)
Lecithinase cleaves lecithin found in egg yolk, releasing insoluble fat (diglyceride) that produces an opaque zone around the colony.
C. perfringens
Describe the principle and application of the test.
Sodium Polyanethol Sulfonate Disk (SPS)
(SPS) disk aids in the identification of anaerobic gram-positive cocci.
An SPS-sensitive, gram-positive anaerobic coccus can be presumptively identified as
Peptostreptococcus anaerobius
An SPS-resistant, spot indole– positive, gram-positive anaerobic coccus can be presumptively identified as
Peptoniphilus asaccharolyticus
Name 2 rapid anaerobic identification panels.
- RapID ANA
- API 20 A
Discuss antimicrobial susceptibility testing considerations for anaerobic bacteria.
Beacuse the isolation, identification, and susceptibility testing of anaerobes is slow, when physicians suspect an anaerobic infection, they routinely select a broad-spectrum agent for empiric therapy that will cover most anaerobes, pending outcome of the culture.
Carbapenems, metronidazole, and the β-lactam combination antibiotics continue to have activity against most anaerobes and are used as empiric therapy.
When should susceptibility testing be performed on anaerobes?
When it is recovered as a pure culture in a sterile site.
- Brain abscess * Endocarditis * Infection of a prosthetic device or vascular graft * Joint infection * Osteomyelitis * Bacteremia
A test for beta-lactamase production VIA cefinase is disc is performed on what type of anaerobic organisms?
Anaerobic GNB (other than B. fragilis group)
What method(s) are used to perform anaerobic susceptibility testing?
- E-test
- Microbroth dilution panels
Other than susceptibilty testing, how can anaerobe-associated diseases be treated?
- Surgery
- Hyperbaric oxygen therapy
- Antitoxins
- Refractory CDAD can be treated with fecal microbiota transplant
What are some indications that anaerobes may be present in a Culture?
- A foul odor on opening an anaerobic jar or bag.
- Colonies present on the anaerobically incubated blood agar plates but not on the CO2-incubated blood plate
- Good growth (> 1 mm in diameter) of black colonies on a BBE agar plate, characteristic of members of the B. fragilis group
- Double zone of hemolysis on CDC (ANO2) suggestive of C. perfringens
Presumptively Identify the Gram-Positive Anaerobes
- Colony morphology: Large, flat colonies; barnyard odor, chartreuse fluorescence
- Gram stain: Thin rods, rare spores
Clostridium difficile
Presumptively Identify the Gram-Positive Anaerobes
- Colony morphology: Large, irregular-shaped, double zone of β-hemolysis
- Gram stain: Boxcar, large, square rods
Clostridium perfringens
Presumptively Identify the Gram-Positive Anaerobes
Colony morphology: Smoothly swarming
Gram stain: Thin rods, subterminal spores
Clostridium septicum
Is often a marker for gastrointestinal cancer
Presumptively Identify the Gram-Positive Anaerobes
Colony morphology: Smoothly swarming but slow growing
Gram stain: Swollen terminal spores
Spot Idole: Positive
Clostridium tetani
Which anaerobes are most commonly associated with a gram stain of gram positive cocci?
Peptostreptococcus spp., Finegoldia, or Peptoniphilus
What anaerobe has a gram stain of Gram negative cocci?
Viellonella spp.
What anaerobe has a gram stain of “fusiform” (thin with pointed ends) GNB and colonies that resemble “breadcrumbs”?
Fusobacterium nucleatum
What anaerobic species has a gram stain of branching, filamentous, non-spore forms gram positive rods?
Actinomyces spp.
Which species of Actinomyces form “molar tooth” colonies?
Actinomyces israelii
Describe Actinomycosis
Actinomycosis is a chronic, granulomatous, infectious disease characterized by the development of sinus tracts and fistulae, which erupt to the surface and drain pus that may contain so-called sulfur granules, dense clumps of bacteria that may be colored.
Actinomyces has a similar gram stain to Nocardia. What stain could be used to differentiate the two?
AFB stain. Nocardia is Acid fast postive. Actinomyces is acid fast negative.
Which anaerobe has a gram stain of non-spore forming “spidery” gram positive bacilli and the colonies described as white and “puffy”?
Cutibacterium acnes
What reactions does Cutibacterium acnes have to spot indole and catalase?
Positive for both
Cutibacterium acnes is a slow growing organism, typically incubated for 10-14 days, and is associated with what type of infection?
Prosthetic joint infection
What organism is associated with IUD (intrauterine device) infections?
Actinomyces
How long should a culture be incubated for when trying to rule out Actinomyces or Cutibacterium acnes?
10-14 days
What anaerobe has a gram stain of pleomorphic GPB that looks like a “dog biscuit”?
Bifidobacterium