Anaerobic bacteria

Laboratory diagnosis in infections produced by anaerobic bacteria

*Definition of terms: bacterial growth in relation with respiratory

processes (use of O2, CO2)

• Obligate aerobes need oxygen because they cannot ferment or respire anaerobically (e.g. Mycobacterium tuberculosis)

• Obligate anaerobes are poisoned by oxygen

• Facultative anaerobes can grow with or without oxygen (e.g. Staphylococcus, Streptococcus, E.coli)

• Microaerophiles need some amount of oxygen but are poisoned by high concentrations of oxygen (e.g. Campylobacter, Helicobacter, Neisseria gonorrhoeae)

http://en.wikipedia.org/wiki/Obligate_aerobe

http://en.wikipedia.org/wiki/Obligate_anaerobe

http://en.wikipedia.org/wiki/Facultative_anaerobes

(Obligate) Anaerobic bacteria- general aspects & definition of terms -

• Energy generated exclusively by anaerobic fermentation (does not generate superoxide radicals = O2 anions)

• Can only grow in the total absence of O2;

• WHY?: Anaerobic bacteria lack the enzymes:– Superoxide dismutase (SOD) – Catalase

(Obligate) Anaerobic bacteria- general aspects & definition of terms - continued

• Superoxide dismutases - antioxidant factors; enzymes which catalyze the dismutation (partitioning) of the superoxide radical (O2 anion) into:– Molecular O2 or – Hydrogen peroxide (H2O2)

• Superoxide = by-product of O2 metabolism; high cellular toxicity

• Hydrogen peroxide = less toxic; degraded by catalase

(Obligate) Anaerobic bacteria- general aspects & definition of terms - continued

CONCLUSIONS: • fermentation in the presence of O2 → superoxide

radicals – toxic if not dismuted (partitioned) by superoxid dismutase into O2 / H2O2;

• furthermore H2O2 – toxic if not decomposed by catalase

• Anaerobic bacteria lack both enzymes (superoxid dismutase and catalase) → mandatory absence of O2 in order for anaerobic bacteria to avoid toxic effects of superoxide radicals and/or H2O2

(Obligate) Anaerobic bacteria- general aspects & definition of terms -

”Friends or Foes?”

• Colonize the human body • involved in the balance of the normal microbial flora:

skin, oropharynx, gastro-intestinal tract, uro-genital tract (urethra, vagina)

• Cause severe infections (endogenous and exogenous)

Obligate Anaerobic Bacteria- Collection and transport of specimens -

• Inoculation asap (within 10 minutes) due to toxicity of atmospheric O2

• Transport: anaerobic tubes with transportation media e.g. modified Cary Blair, Stuart– minimal nutrients to increase survival of organisms without

multiplication – sodium thioglycollate - to provide low oxidation-reduction

potential – alkaline pH – to minimize bacterial destruction by acid

production– phenol red indicator (red at alkaline pH, yellow at acidic pH)– Redox indicator: resazurin – turns pink in the presence of O2

Obligate Anaerobic Bacteria- Collection and transport of specimens -

continued“Hungate tubes”:• Disposable/autoclavable

screw thread style tube designed to maintain anaerobic culture conditions

• butyl rubber stoppers,• screw cap 9 mm opening

Obligate Anaerobic Bacteria- Macroscopic and microscopic exam -

• Suggestive signs of anaerobic infection:– Fetid odour– Purulent aspect– Necrotic tissues

– Gas (e.g. ”gas gangrene” – wound infected by Clostridium perfringens)

• Microscopy: Gram stained smears (methanol fixation to preserve cellular elements)

Obligate Anaerobic Bacteria- Isolation -

• Nonselective media: blood agar

• Selective media requirements (CLSI: Clinical Laboratory Standards Institute): – 3 days of strict anaerobic

incubation → mandatory growth of:

– Bacterioides fragilis, Clostridium perfringens + Fusobacterium nucleatum, Peptostreptococcus anaerobius, Bacteroides levii

Obligate Anaerobic Bacteria- Identification -

• API 20 A Identification of anaerobes in 24-48 hours

• Fermentation tests, which are the reference tests for the identification of anaerobes

• Easy-to-use: suspension prepared directly in the API 20 A medium.

• Polyvalent system for all anaerobes, both Gram (+) and Gram (-)

Obligate Anaerobic Bacteria- Human infections -

Exogenous sporulating• Gram positive bacilli:• Genus Clostridium:

– Gas gangrene clostridia– Clostridium tetani– Clostridium botulinum– Clostridium difficile* (may

cause both exogenous and endogenous infections)

Endogenous nonsporulating• Gram negative bacilli:

– Family Bacteroidaceae:• Bacteroides, Prevotella,

Prophyromonas, Fusobacterium

• Gram positive bacilli: – Propionibacterium acnes– Actinomyces israelii

• Gram positive cocci:– Peptococcus,

Peptostreptococcus• Gram negative cocci:

– Veillonella

Bacterial survival outside host

Spores: reproductive structures adapted

for longtime survival in unfavourable

conditions

(etymology: ancient Greek spora = seed)

Bacterial spores - outer layer of keratin resistant to chemicals, staining and heat → bacterium able to stay dormant for years, protected from temperature differences, absence of air, water and nutrients

Spore forming bacteria:

• Genus Clostridium;

• Bacillus spp (B. anthracis).

Genus Clostridium- Clinical significance -

• Exogenous infections: – Gas gangrene – Tetanus– Botulism– Food poisoning

• Endogenous infections: • Clostridium difficile

Definition of terms: Gangrene

• tisular death (necrosis) caused by lack of blood supply (= absence of O2 and nutrients in the respective area of the

organism)• potentially life-threatening condition • may occur by:

– injuries, trauma (compression of blood vessels)– frostbite (freezing of exposed extremities)– infection– chronic diseases affecting blood circulation e.g. diabetes

Gas gangrene clostridia

• Gas gangrene – severe invasive infection starting from infected wounds, rapid systemic invasion

• Caused by: Clostridium perfringens, + Cl. oedematiens, Cl. histolyticum – found in water, soil air + intestinal comensals

• Severity augmented by toxin production

• Collection of specimens: profound wound secretion, tissue fragments

Gas gangrene: Clostridium perfringens

• Oedema, necrosis, large blisters, crepitation

• Lower image: large incision for oxygen exposure of infected tissues

Gas gangrene clostridia- Microscopic examination -

Gram stained smear: • total absence of cells (no

PMNs, no epithelial cells, etc)

• short, thick, Gram positive bacilli

• no spores (no spore forming in vivo)

Gas gangrene clostridia: Cl. perfringens- Isolation and identification -

Blood agar: double hemolysis

Gas gangrene clostridia: Cl. perfringens- Isolation and identification - continued

Egg yolk agar (EYA): enriched

medium for presumptive id of

anaerobes e.g. Clostridium • Egg yolk suspension: detection

of enzymes:– lipase (iridescent sheen on

colony surface) and – lecitinase (opaque precipitate

around colonies)

Gas gangrene clostridia: Cl. perfringens- Isolation and identification - continued

• Reverse CAMP test Principle: synergistic effect between Streptococcus agalactiae (group B) and hemolytic Clostridium perfringens

• Initially intended as an improvement of CAMP test i.e. replacement of S.aureus by Clostridium perfringens for the identification of Streptococcus agalactiae (Group B)

• Then the idea comes up to use Streptococcus agalactiae (group B) to identify Clostridium perfringens (= the reverse CAMP test)

The CAMP test: id of Streptococcus agalactiae

(A) Streptococcus (group B) positive test (enhanced hemolysis)

(B) Streptococcus pyogenes (group A) negative test

(C) Staphylococcus aureus – replaced by Clostridium perfringens in reverse CAMP test (see next slide)

The reverse CAMP test

Identificationof Clostridiumperfringens: (A) Reverse CAMP-

positive Clostridium perfringens (”bow tie”)

(B) reverse CAMP-negative Clostridium septicum streaked at right angles to

(C) Streptococcus agalactiae (group B)

Clostridium perfringens- Antimicrobial susceptibility -

• Sensitivity to: penicillin G, erythromycin, ampicillin, metronidazole

• Natural resistance to tetracyclines











– Veillonella

Clostridium tetani

• Habitat: intestinal tract of animals (sheep, cattle); vegetative bacteria eliminated with faceces; contamination of soil (spore formation)

• Infection occurs via:– Skin lesions contaminated with spores e.g. wound highly

contaminated with dirt, dust; extensive wounds with crushed tissues and foreign bodies (accidents);

– spores germinate into vegetative bacteria which multiply at the entry and produce tetanic toxin (disseminated) – 2 components:

• Tetanospasmin – muscle spasms• Tetanolysin – cardiotoxic

Clostridium tetani

Clinical significance:• Tetanus = Generalized tetanus (most comon form):

– onset with trismus (spasms of the face and chewing muscles popularly called “lockjaw”→ characteristic facial expression risus sardonicus or sardonic grin);

– further evolution: swallowing becomes increasingly difficult; severe spastic hyperextension of head, neck and spine (opisthotonos)

(effects of the tetanus exotoxin: tetanospasmin)

• Lethal outcome ~ 1 in 10 cases - spastic paralysis of respiratory muscles

Left: Risus sardonicus (rigid facial grin)Right: Opisthotonos (spastic contraction with

hyperextension of head, neck and spine)

Tetanus

• Vaccine preventable disease: several vaccines used to prevent tetanus among children, adolescents, and adults; e.g. combined vaccines against diphteria, pertusis and tetanus (e.g. DTaP) or tetanus and diphteria (e.g.TD) – Immunization schedules

• In Romania: tetanus containing vaccines given at the ages of:– 2, 4, 6, 13 months, 4 years (DTP) + 14 years (dT) +– (recommended) dT every 10 years

Definition of terms: Immunization schedule

• series of vaccinations, including the timing of all doses, which may be either recommended or compulsory,

depending on the country of residence • Examples:• http://www.cdc.gov/vaccines/schedules/hcp/child-adolescent.html#printable

• http://www.nhs.uk/Conditions/vaccinations/Pages/vaccination-schedule-age-checklist.aspx

• http://en.pediatricblog.info/2011/02/romanian-mandatory-vaccination-schedule.html

http://en.wikipedia.org/wiki/Vaccination

http://www.cdc.gov/vaccines/schedules/hcp/child-adolescent.html

http://www.nhs.uk/Conditions/vaccinations/Pages/vaccination-schedule-age-checklist.aspx

http://en.pediatricblog.info/2011/02/romanian-mandatory-vaccination-schedule.html

Tetanus prophylaxix in routine wound management

1st step: Assess wound - Clean, minor wound: • Q1: Has patient completed a primary tetanus-diphteria

series? (= minimum 3 doses of tetanus- and diphteria containing vaccine: e.g. at 2, 4 and 6 months of age)– NO/Unknown: Administer vaccine today (i.e. complete series per

age-appropriate vaccine schedule)– YES→Q2: Was the most recent dose within the past 10 years?

• NO: Administer vaccine today (next dose per age-appropriate schedule)

• YES: vaccine not needed today; next dose will be given at 10 years after the last dose

Tetanus prophylaxix in routine wound management - continued

1st step: Assess wound: “tetanigenic potential”:contaminated with dirt, faeces, saliva, soil; puncture wounds (lack of O2 in profound layers of wound); animal bites, burns, frostbite

• Q1: Has patient completed a primary tetanus-diphteria series?– NO/Unknown: vaccine + tetanus immune globulin (TIG) today– YES→Q2: Was the most recent dose within the past 5 years?

• NO: Administer vaccine today (next dose per age-appropriate schedule)

• YES: Vaccine not needed today (next dose at 10 years from last dose)

Clostridium tetani

• Laboratory diagnosis only required in suspicion of iatrogenic infections e.g. infection of umbilical cord stump, post-partum infections, etc

• In most cases diagnosis relies on clinical aspect and history (tetanigenic circumstances e.g. wounds contaminated with dirt, faeces, saliva, soil; puncture wounds; animal bites, burns, frostbite)

• IMPORTANT FACTS:– no human to human transmission– Vaccine preventable











– Veillonella

Clostridium botulinum - Clinical significance -

Foodborne Botulism: • Ingestion of botulinic toxin

contained in canned vegetables, fish, meat (toxin production generated gas → can lid appears bulging/swollen)

• Toxin resists gastric acidity, enters the intestine → lymph vessels → blood stream→ acts on nervous system: progressive flaccid paralysis

Wound botulism: i.v. / intradermic drug users

• Possible use as biological weapon

Clostridium botulinum - Clinical significance - continued

• Onset symptoms: double vision, blurred vision, drooping eyelids, slurred speech, difficulty swallowing, dry mouth, and muscle weakness

• if untreated: descending flaccid paralysis; death by paralysis of respiratory muscles

• Treatment: botulinum antitoxin – asap!!

Clostridium botulinum- Laboratory diagnosis -

• Performed in reference laboratories

• Specimens: blood for serology, vomit, faeces, suspected food

• Identification based on:– Gram stain: Gram positive

bacilli + spores; – anaerobic growth; – neurotoxin detection:

experimental disease in mice; immunoassay; molecular techniques











– Veillonella

Clostridium difficile- Clinical significance -

• Pseudomembranous colitis: bloating and severe diarrhoea

• Endogenous: bacteria replaces normal intestinal flora that has been compromised, usually following antibiotic treatment for an unrelated infection; C. difficile gains a growth advantage (positive selection) and overruns the intestinal microbiome; “antibiotic-associated diarrhoea”

• Exogenous: accidental ingestion of spores e.g. incomplete/incorrect hospital management of infected patient (isolation, disinfection, etc) leads to spore contamination of objects → spore ingested by another patient (when prevention guidelines are not strictly followed e.g. hand washing, cleaning, PPE, disinfection)

Clostridium difficile- Prevention guidelines in clinical settings -

Examples:• http

://www.documents.hps.scot.nhs.uk/about-hps/hpn/clostridium-difficile-infection-guidelines.pdf

• http://www.idsociety.org/uploadedFiles/IDSA/Guidelines-Patient_Care/PDF_Library/cdiff2010a.pdf

• http://d2j7fjepcxuj0a.cloudfront.net/wp-content/uploads/2013/04/ACG_Guideline_Cdifficile_April_2013.pdf

http://www.documents.hps.scot.nhs.uk/about-hps/hpn/clostridium-difficile-infection-guidelines.pdf



http://www.idsociety.org/uploadedFiles/IDSA/Guidelines-Patient_Care/PDF_Library/cdiff2010a.pdf

http://d2j7fjepcxuj0a.cloudfront.net/wp-content/uploads/2013/04/ACG_Guideline_Cdifficile_April_2013.pdf











– Veillonella

Endogenous nonsporulating bacilli

Gram positive • Propionibacterium acnes:

involved in juvenile acne, blepharitis together with staphylococci, corynebacteria; morphology similar to corynebacteria

• Actinomyces israelii: comensal flora of the oral cavity; involved in periodontal disease, abscesses (in immune compromised patients)

Gram negativeBacteroides, Prevotella,Prophyromonas,

Fusobacterium- Normal flora- Isolation in naturally

sterile sites – always pathological

Endogenous nonsporulating cocci

Gram positive• Peptococcus,

Peptostreptococcus – normal oral flora; may be involved in infections together with other anaerobes (e.g. skin infections after human bites)

Gram negative• Veillonella – normal oral

flora; may be involved in purulent alveolar infections

Education

Anaerobic bacteria