HAD 381 - Aerobic Gram-Positive Rods

Spore forming bacilli

• Bacillus spp

Non-spore forming bacilli

• Morphologically regular

  • Listeria, Erysipelothrix, Lactobacillus, Kurthia

• Coryneform or irregular

  • Corynebacterium (from Green koryn club)

• Nocardioforms (filamentous) and aerobic actinomycetes

  • Nocardia, Streptomyces

Spore forming gram-positive rods: Bacillus spp.: Most pathogenic:

• B. anthracis (anthrax)

Spore-forming gram-positive rods: Bacillus spp.: Most frequently isolated in CML:

• Many species found as contaminant

• B. cereus: “Fried rice syndrome”

  • Emetic toxin

• B. subtilis: opportunistic infection

How does anthrax work:

1. Anthrax spores are inhaled

2. Anthrax spores enter lungs and travel to alveolar spaces

3. Spores are transported through the lymph system to the mediastinal lymph nodes where they make toxins that are deadly

The Anthrax Cycle:

• Vegetative Forms: Bacteria in animal waste and decomposition (Cattle: Inactive, organism cannot replicate)

  • When exposed to oxygen, will become anthrax spores — Converts inactive form to active

• Can be cutaneous from open wounds/abrasions or infected animal (ex. fly)

• Can be inhaled through respiratory complications

• Can be ingested

Anthrax:

• Primarily a disease of cattle, sheep, horses, and goats

• Humans become infected incidentally when brought into contact with diseased animals, which includes their flesh, bones, hides, hair and excretement

  • BIOTERRORISM AGENT

Cutaneous anthrax:

• Most common acquired via injured skin or mucous membranes (95%)

Gastrointestinal anthrax:

• Analogous to cutaneous anthrax but occurs on the intestinal mucosa

Inhalation anthrax:

• Wool sorters’ disease

  • Pulmonary, rare

  • Workers who handle animal hide are more susceptible

Anthrax Toxin Subunits:

• 2 A subunits

• 1 B subunit

• All together create “holotoxin” which apparently random mixtures of A subunits

A subunits:

• Edema factor (adenylate cyclase)

• Lethal factor protease

Edema factor (adenylate cyclase):

• Converts ATP → cAMP which causes activity of cell such as imbalance of fluid, which causes accumulation of fluid

Lethal factor protease:

• Receptor attaches to cell and will kill macrophage to survive

B subunit:

• Protective antigen

Anthrax Virulence:

• poly-D-glutamic acid capsule

• tripartite toxin

  • Protective antigen (PA) : B subunit

  • Edema factor (EF) : A subunit

  • Lethal factor (LF) : A subunit

• The three proteins combine in pairs to produce the lethal factor (PA+LF) ad edema factor (PA + EF)

Bacillus spp. BAP

• Aerobic or faculative anaerobic

• Large (4-5 mm), flat, spreading, grey white colonies, with irregular margins

• Many beta hemolytic (Except B. anthracis which is non hemolytic)

• Gram stain of Bacillus anthracis, showing endospores

• Large rectangular rods, form chains

• Spores do not cause swelling of cells and perfectly fit inside

How to differentiate from Bacillus spp. gram stain/microscopic wise?

• Malachite green/safranin spore staining

  • Malachite green stains spore green

  • Safranin stains rods

Differential tests for B. anthracis

• Hemolysis negative

• Motility negative

• Gelatinase negative

• Salicin fermentation negative

• Penicillin susceptible

• Other Bacillus spp. are positive and resistant (except B. subtilis which is susceptible to penicillin)

Bacillus cereus clinical significance:

• Associated with gastrointestinal infections, local infections, and systemic infections

• Specimens for isolation

  • Blood and CSF (systemic infection)

  • Sputum and Pleural Fluid (pneumonia)

  • Wound, eye, bone marrow, joint fluid (Local infections)

  • Feces and suspect food (foodborne infections)

    • Submitted to public for analysis

Spore-forming rods:

• Aerotolerant Clostridium

  • Clostridium tertium and Clostridium perfringens are examples

    • Grow best in anaerobic environment, but may grow in atmosphere with oxygen

    • General clue is initial sparse growth of tiny colonies compared to the growth on anaerobic media/conditions displaying much more growth and larger colonies

  • Gram stain morphology resembles Bacillus species (large, with or without spores)

How to differentiate between Bacillus spp. and Clostridium spp.

• Most Clostridium are catalase negative and form spores under anaerobic conditions

• Bacillus spp. are catalase positive and form spores under aerobic conditions

Species of Corynebacterium other than C. diphtheriae

• Listeria, Erysipelothrix, Kurthia are coryneform organisms too

• Generally considered contaminants when encountered in cultures of clinical material

• Inhabit the skin and mucous membranes of the upper respiratory tract, urethra, and vagina (normal flora)

• Erysipelothrix can cause infections in otherwise normal persons (immunocompetent)

• Listeria can cause infections in both normal hosts, but mostly immunocompromised persons

• Kurthia has been rarely isolated in cases of endocarditis

Diphtheroid infection has been associated with?

• Vascular prosthesis or immunosuppression

Coryneform or diphteroid bacteria morphology

• Irregular shape (pleomorphic)

• Arranged in V forms or palisades (non-branching)

• “Chinese-letter” or “picket fence” arrangement

• Stains unevenly (metachromatic)

  • This uneven stain may lead to beaded appearance

  • May need to report as gram variable if staining unevenly

Corynebacterium spp.

• Non-spore forming

• Non-motile

• Catalase positive

• Ferment glucose and mannitol

• No H2S gas production

• Non-acid fast

Corynebacterium diphtheriae

• Causes diphtheria

  • Potentially fatal illness that kills 5% to 10% of infected persons

  • Affects the mucous membranes of the nose and throat: forms pseudomembrane

  • Can also affect the skin by infecting existing open wound": cutaneous

  • In advanced stages, toxins reach circulation and can damage the heart and nervous system: systemic infection

• Isolated infrequently in developed countries since vaccination has been implemented, but still a problem in underdeveloped or developing nations

Diphtheria infection in oropharynx

• Causes a strong inflammation and formation of pseudomembrane that can lead to respiratory obstruction (suffocation)

• Produce a very powerful exotoxin which spreads by the bloodstream affecting heart muscle and nerve endings: can cause paralysis and is systemic

Diptheria toxin and diphtheria toxin receptor:

• Toxin gene encoded by a bacteriophage

• Toxin is a single polypeptide chain consisting of two subunits linked by disulfide bridges, known as A-B toxin

• Binding to the cell surface of the B subunit allows the A subunit to penetrate the host cell and block protein synthesis by transfer of ADP-ribose from NAD to a diphthamide residue of EF-2

Diphtheria Toxin:

• Synthesized by toxigenic strands of Corynebacterium diphtheria

• Toxin enters through receptor mediated endocytosis

• Acidification of endocytic vesicle allows A to dissociate from B and then enter the cytoplasm

C. diphtheriae macroscopically

• Grow in BAP (common medium for nasopharingeal specimens)

• Colony type depends on biotype:

  • Biotype Intermedius: Small gray or translucent

  • Biotypes mitis, belfanti and gravis: White opaque, larger

• Non hemolytic or alpha hemolytic

• Grow best at 37 C on a blood or serum containing medium (Loeffler’s serum or serum tellurite medium)

• Facultative anaerobes

• Can grow in ambient air or may be incubated in a 5-10% CO2-enriched environment

Serum tellurite medium

• Also known as Cystine-tellurite or Tinsdale medium

• Black colonies indicated tellurite reduction

• Selective medium when C. diphtheriae is suspected

• Inhibits normal flora from the nasopharingeal tract

Loeffler’s medium:

• Used to enhance granule formation before Methylene Blue staining, Alkaline Blue or Albert’s and Neisser’s (Not differential or selective)

Characteristics of Loeffler’s medium:

• Volutin (metachromatic) dark purple granules composed of inorganic polyphosphates (volutin) giving the rods a beaded appearance when stained with Neisser or Albert stains

C. urealyticum

• Causes UTI

• Multi-drug resistant

• Lipophillic

• Urea positive

• Urinalysis may reveal alkaline urine and struvite crystals (Presence of struvite crystals) magnesium ammonium phosphate due to urease activity

• Hold urine cultures more then 24 hours to obtain isolation - slow growing

C. jeikenium

• Multidrug resistant — can be fatal, esp to immunocompromised

• Cause of endocarditis, septicemia, foreign body infection

C. pseudodiphtherium

• Normal flora

• Can cause pneumonia

C. equi

• Now Rhodococcus equi

• Found in soil, causes diseases in horses and goats

• In immunocompromised cause TB-like infections (mycobacterium)

Arcanobacterium general characteristics

• Short pleomorphic GPR

• Catalase negative

A. pyogenes and A. haemolyticus

• Beta-hemolytic (narrow)

• May be PYR positive

A. haemolyticum

• CAMP inhibition positive

• Xylose and gelatin negative

A. pyogenes and A. bernardiae

• CAMP inhibition negative

• Xylose and gelatin positive

A. pyogenes

• ONPG positive

Regular rods:

• Non-spore forming, gram positive rods which have two parallel sides with rounded ends

  • Includes Listeria spp. Lactobacillus, and Erysipelothrix

• Colony morphology similar to other microorganisms, so the gram stain is important role in their identification

Listeria spp.

• Non-spore forming, Gram positive bacilli

• Facultative intracellular anaerobe

• Catalase positive and oxidase negative

• Colony and Gram stain morphology may resemble Group B streptococci

Main pathogenic species of Listeria

• Listeria monocytogenes

  • Food poisoning (listeriosis)

  • Can cause outbreaks invasive listeriosis

  • Affects pregnant women, their babies (neonatal meningitis), the elderly and immunosuppressed adults

Other rare human pathogen of listeria:

• L. ivanovii

Listeria monocytogenes virulence factors:

• Intracellular pathogen

• Replicates in pathogen

• Actin binding protein (movement inside cytoplasm)

• Phospholipase C and Listeriolysin O (lysis of the vacuole)

Listeria Monocytogenes laboratory tests

• Grow well on a wide variety of laboratory media

• Optimal growth 30-35C

• Grow well at 4C (selective condition)

• Beta hemolytic (other Listeria spp are non-hemolytic — differential)

• Catalase positive (Strep is negative)

Listeria monocytogenes macroscopically

• Small

• Beta hemolytic

• May have blue/green sheen

  • Narrow zone of beta-hemolysis

• Resembles GBS and may react with Streptococcal Group B and G antisera

• CAMP positive

L. monocytogenes microscopically

• Coccobacilli

• Single or short chains

• No branching

• Can form filament in older cultures

• Can be mistaken by streptococci (GBS also cause neonatal meningitis)

Listeria monocytogenes motility:

• Non motile at 35C

• Motile at 25C "

  • “Umbrella” type or Xmas tree shape

• Tumbling motility (end-over-end) in hanging drop preparations

  • Hanging drop done with concave microscope slide and you put drop of organism on coverslip and put the the overslip on top, upside down

Important L. monocytogenes: ID tests

• CAMP test positive (GBS also CAMP+)

• Non motile at 35 C (like GBS) motile @25C

• Ferments glucose, trehalose, salicin

• Esculin hydrolysis: positive

  • Used in differential media, black colonies

Erysipelothrix spp.

• E. rhusiopathiae causes economically important disease in swine called erysipelas (different from erysipelas that is caused by GAS) also in lambs, calves, birds, and fish

• Humans become infected (rare) through exposure to infected or contaminated animal or animal products

• Occupational disease: affects veterinarians, abattoir workers and fisherman (“fish handlers’ disease”) form of skin infection in the fingers (eryispeloid) seal finger or whale finger

• Occasionally cause septicemia and endocarditis

E. rhusiopathiae

• Non sporing gram positive bacilli or long branching filaments

• Might look like gram-negative (decolorize easily)

Erysipelothrix rhusiopathie macroscopically/lab characteristics

• Non-motile

• Facultative anaerobe

• Improved growth @ 5-10 % CO2

• On BAP two distinct colony forms (non-hemolytic)

  • Smooth: small convex and circular, transparent glistening colonies

  • Rough: large, flat opaque

Erysipelothrix rhusiopathie ID tests

• Catalase neg

• Oxidase neg

• Indole neg

• Urea neg

• Esculin neg

• Motility neg

  • TSI K/A, with H2S

    • (as opposed to L.monocytogenes)

  • Weakly fermentative (glucose +)

  • Gelatin stab at 25C (Not used for gelatinase, but growth pattern)

  • Growth": pipe cleaner or test tube brush pattern (Gelatinase -)

Lactobacillus

• Normal microbiota of vagina, gastrointestinal tract, and oropharynx

• Widely distributed in nature

• Make up 90% of the normal human vaginal flora

• Also normal flora of mouth and GI tract

• Facultative anaerobe

• Part of the lactic acid bacteria group

  • Convert lactose into lactic acid

• Opportunistic infections

  • Bacteremia, endocarditis, meningitis (rare)

Regulators of the vaginal ecosystem

• Lactobacillus

• May prevent urogenital infections by

  • Production of lactic acid, bacteriolicins (toxins produced by bacteria to inhibit growth of other bacteria), and hydrogen peroxide

  • Competitively excluding attachment and vaginal colonization by other microorganisms

• Lactobacillus sp. adhering to a vaginal epithelial cell as seen on a Gram stain of vaginal fluid (x 1,000 magnification)

• 60-70% covered in lactobacillus, which is normal

  • No lactobacillus, could be indicitive of BV

  • Coccobacilli also indicative of BV

Lactobacillus spp. microscopic morphology

• Long, slender gram positive pleomorphic bacilli

• Non-spore forming

Lactobacillus lab diagnosis results

• Catalase negative

• Oxidase negative

• Nitrate negative

• Indole negative

• H2S negative

• Gelatinase negative

Kurthia spp.

• Associated with endocarditis, pneumonia, and bacteremia (patient must be seriously immunocompromised to acquire)

• Regular, unbranched, relatively large GPB

• Rounded ends, occur in chains

• Motile by peritrichous flagella

• Non-acid fast

• Catalase positive

• Oxidase negative

Irregular Rods

• Gram positive rods which share pleomorphic microscopic morphology

  • Gardnerella

  • Aerotolerant Actinomyces

  • Actinomycetes

  • Nocardia

Gardnerella

• Best medium for growth is human blood bilayertween (HBT) agar; V agar with human blood is also used for isolation

• Beta hemolytic on HBT (Bc lots of normal flora in vaginal environment)

• Capnophilic

• Inhibited by Sodium polyanethole Sulfonate (SPS) → anticoagulant that prevents immune cells from attacking organism that are present

Gardnerella clinical significance

• Normal flora

• Loss of lactobacillus and predominance of Gardnerella and anaerobic GNR are associated with bacterial vaginosis

• Linked to maternal and neonatal infections

Gardnerela vaginalis

• A corynebacterium-related species (previously C. vaginalis)

• Causes bacterial vaginosis (BV)

  • Different from vaginitis (Acquired from candida)

• Typically associated with other bacteria

• Bacteremia (rarely)

• Common in asymptomatic women (70%) and girls (14%)

• Also found in male urethra

Gardnerella identification:

• Most laboratories presumptively identify using Gram stain, catalase, and colony morphology

Gardnerella microscopic morphology:

• Gram-variable rod: Could stain as gram negative or gram positive because peptidoglycan layer is much thinner than the Corynebacterium, Lactobacillus and Staphylococcus

Gardnerella Colony morphology:

• Tiny, white to gray colonies

• Non-hemolytic on BAP

• Appears after 48-72 hours of incubation

Bacterial Vaginosis: Clue cells

• Many labs use Gram stain to evaluate the presence or absence of bacterial vaginosis

• In these smears, vaginal epithelial “clue cells” will be covered with bacterial morphotypes. Sheer number of organism cover the margin of epithelial cells

Gardnerella Key Biochemical Reactions:

• Hippurate and starch positive

• Alpha-glycosidase positive and beta-glucosidase negative (enzymes present in cleavage of diff bonds present in carbohydrates)

• Raffinose negative (carbohydrate)

• Inhibited by SPS

Whiff test:

• A drop of 10% KOH mixed with vaginal fluid and production of fishy smell of indicative of positive whiff tests and bacteria vaginosis

Gardnerella vaginalis susceptibily

• Metronidazol, triemetroprim ad sulfonamide

  • Differentiates from other catalase negative, coccobacillus (e.e. lactobacillus)

Nocardioforms (forms in brain tissues) and aerobic actinobacteria (previously known as Actinomycetes)

• Fungus like bacteria

• Aerobic, facultative anaerobes or obligate anaerobes

• Branched

• Filamentous hypahe

• Reproduction

  • By producing spore

  • By fragmentation of the hyphae

Aerotolerant Actinomyces general characteristics

• Mykes Greek for “fungus”

• Exhibit true branching

  • Non spore-forming

  • Non-acid-fast

  • Facultative anaerobes

  • Capnophilic

  • Grows best anaerobically

Aerotolerant Actinomyces Identification: Microscopic morphology

• Irregular staining, pleomorphic, GPR

• Diphterodial and branching rods common

• Microscopy smears may show filaments with branching

Aerotolerant Actinomyces macroscopically

• Microcolonies form after 18 to 24 hours incubation

• “spider” or granular centered colony with peripheral branching filaments

• Some form colonies that are smooth without branching filaments

• Abscess drainage may contain visible yellow to orange sulfur-like granular colonies

Aerotolerant Actinomyces Key biochemical reactions

• Most are catalase negative

• Non motile

• Fermenters

• H2S positive

• Gelatin negative (straight line growth)

  • Additional biochemical testing (traditional methodology or rapid/commercial kits) can be performed if speciation is necessary including testing

Other rare human pathogen of Listeria:

• L. ivanovii

Aerotolerant Actinomyces clinical significance:

• members of the commensal or normal flora in various

  body sites, especially the mouth.

• They are the causative agent of actinomycosis, which causes abscesses, often of the jaw, which drain via sinus

  tracts to the skin surface. Lumpy jaw

Aerobic Actinomycetes general characteristics:

• Group of GPR that form thin, beaded, branching filaments.

• Some extend filaments into the air to form aerial mycelium

• Some are partially acid-fast

• May form microcolonies

• Infrequent isolates

Aerobic Actinomycetes useful biochemical tests:

• Lysozyme resistance

• Substrate decomposition of Casein, Xanthine, and Tyrosine

• Opacification of middlebrook 7H10 agar

• Gelatin liquefaction

• Acetamide

• Arylsulfatase

• Citrate

• Commercial kits

Nocardia spp general characteristics:

• Posses tuberculostearic acid like Mycobacterium spp. Contain short chain of mycolic acid in their cell wall as all the Gram positive. The presence of mycolic acid

  is standard in all but the length of the chain varies among genera.

• Opportunistic pathogens

• Obligate aerobe

• Some species require cooler temperature for optimum growth

• Most commonly isolated Actinomycete

• Branching, beaded, filamentous bacteria

• Can cause "Sulfur granules" in nocardial mycetomas

• stains acid fast* (weakly)

• Mycobacteria strong acid fast

• Actinomyces spp.non acid fast

• L-forms (cell wall deficient organisms) can survive in macrophages for days, may account for treatment failure

Nocardia microscopic morphology:

• GPR

• Branching or beaded

• Partially acid-fast (Modified Kinyoun stain)

Nocardia colony morphology:

• Wrinkled, dry, chalky, adherent

• White to yellow, orange, tan, or brown

• Beta-hemolytic on SBA

• Selective medium used for Legionella can grow Nocardia

• Observe aerial mycelium and conidia on slide culture

Nocardia key biochemical reactions:

• Speciate with molecular methods

• ID to complex using biochemicals and susceptibility results

• Catalase positive

• Arylsulfatase resistant

• Most are Urease positive

Nocardia clinical significance:

• An immunocompromised individual inhales the organism into their lungs and develops respiratory infections. From there it can disseminate to other parts of the body and be isolated from blood and cerebrospinal fluid.

• 80% of lung infections caused by N. asteroides

• Immunocompetent persons usually develop skin infections due to traumatic injury

  » Mycetoma

  » Lymphocutaneous

  » Superficial skin infection

• 80% of skin infections caused by N. brasiliensis

• Granules resembling the sulfur granules of Actinomyces can be observed in pus

• Can spread from blood to brain, skin, eyes, kidney, joint, bones, and heart

80% of lung infections are caused by?:

• N. asteroides

80% of skin infections are caused by?:

• N. brasiliensis

• Differences between Actinomyces and Nocardia

Nocardia common manifestations:

• cutaneous disease: mycetoma or actinomycetoma

• pulmonary (similar to TB)

• systemic

• CNS disease

N. asteroides complex including N. facinica and N. nova cause?:

• Most serious systemic and CNS diseases

N. brasiliensis causes?:

• Cutaneous disease mostly in tropical countries and southeastern of USA

N. pseudobrasiliensis causes?:

• Systemic infections, including the CNS

Nocardia diagnosis:

• Grow in BAP, Sabouraud, brain heart infusion (BHI)

• prolonged incubation may be required for their detection (up to 2 weeks)

• grow on most nonselective media

• specimens with mixed flora can over grow the nocardia colonies

• Selective media that may increase yield:

  –Thayer-Martin agar with antibiotics

  – Buffered charcoal-yeast extract (BCYE) medium

Streptomyces general characteristics:

• Second most commonly isolated Actinomycete

• Catalase positive

• Requires 2 to 3 weeks incubation for growth

Streptomyces microscopic morphology:

• Gram positive branching bacilli

• Right angle formations

• Nonacid-fast

• Form branching filaments of cells which become a network of strands called a mycelium

Streptomyces: colony morphology:

• “spider” microcolonies

• Glabrous, waxy, and heaped

• Most are grayish-white

• Observe aerial mycelium and conidia on slide cultures